VIROLOGY 180, 135-152 (1991)

Mutations in Conformation-Dependent Domains of Herpes Simplex Virus 1 Glycoprotein BAffect the Antigenic Properties, Dimerization, and Transport of the Molecule

ISHTIAQ QADRI, CONCEPCION GIMENO, 1 DAVID NAVARRO, AND LENORE PEREIRA'

Division of Oral Biology, School of Dentistry, and Department of Microbiology,and immunology, School of Medicine,University of California San Francisco, San Francisco, California 94143

Received May 22, 1990; accepted August 27, 1990

Glycoprotein B (gB) is a component of the herpes simplex virus 1 envelope that is required for penetration of virionsinto cells . We constructed 1 1 mutants in the g13 gene by deleting the carboxy terminus of the molecule, inserting linkersinto the ectodomain and intracellular region, and creating point mutations in cysteine residues . To identify regions ofthe molecule that affect the formation of epitopes on gB, we cloned the mutated genes into a eukaryotic expressionvector, transfected them in C0S-1 cells, and reacted the gene products in immunofluorescence and immunoprecipita-tion tests with a panel of monoclonal antibodies . Our findings are as follows . (i) The ectodomain of gB between residues600 and 690 is highly antigenic and contains residues that specify 8 continuous epitopes and affect the conformation of12 discontinuous epitopes . Residues that form a novel neutralizing domain and affect the assembly of gB dimers arecontained in this region . Dimerization of gB does not require the transmembrane region or the intracellular carboxyterminus. (ii) Transport of the insertion mutants was aberrant and depended on the site mutagenized . Insertions oflinkers at residues 391, 413, and 479 of the ectodomain precluded the binding of neutralizing antibodies that recognizeresidues in four discontinuous-epitope domains ; the latter mutant in intact gB was not translocated to the cell surface .In contrast, insertions at residue 600 of the ectodomain and 810 of the intracellular domain did not affect the conforma-tion-dependent epitopes or gB transport . (iii) Substitution of serines for cysteine residues in a discontinuous-epitopedomain in the midregion of gB altered the conformation of both proximal and distal sites . Seven epitopes were lost bymutagenesis of cysteine 382 and 4 epitopes by mutagenesis of cysteine 334 . Together with previous findings, theseresults indicate that the ectodomain of g8 contains three topographically distinct neutralizing regions, one of continu-ous and two of discontinuous epitopes . The continuous-epitope domains that map at the amino terminus are notaltered by distal mutations . In contrast, the domains of discontinuous epitopes, assembled by juxtaposing residues onthe surface of gB, are affected by proximal and distal mutations that alter the antigenic structure, processing, andsurface transport of gB . o 1991 Academic Press, Inc.

INTRODUCTION

Herpes simplex virus 1 (HSV-1) glycoprotein gB is acomponent of the virion envelope and is one of threeglycoproteins required for viral infectivity. Highly con-served homologs have been identified in the genomesof herpes simplex virus 2 (HSV-2) (Bzik et al ., 1986 ;Stuve et al., 1987), Epstein -Barr virus (Pellett et al.,1 985a), varicella zoster virus (Davidson and Scott,1986), and human cytomegalovirus (Cranage et al .,1986). Studies on patient sera showed that gB is apotent target of humoral immunity in human infections(Eberle and Mou, 1983 ; Eberle et al ., 1985 ; Norrild,1980). When used to immunize animals, immunoaffin-ity-purified gB elicits protective immunity (Meignier etat, 1987) .

Nucleotide sequencing of the HSV-1 (F) gB gene andsecondary structure analysis predicted that gB is a typi-

1 Present address : University of Valencia, Facultad De Medicirta,Valencia, Spain .

To whom requests for reprints should be sent .

135

cal transmembrane glycoprotein, 874 amino acids inlength, with a 29-amino-acid cleavable signal se-quence (Pellett et at, 1 985b) . The glycoprotein com-prises three domains: an amino-terminal hydrophilicextracellular domain of 696 amino acids, a hydropho-bic transmembrane domain of 69 amino acids, and acharged carboxy terminus of 109 amino acids that isintracellular. Analysis of the amino acid sequence ofHSV-1 strain KOS predicted several differences in thesequence of residues, particularly within the amino ter-minus of the molecule (Bzik etal., 1984b;Kousoulasatal., 1988) . HSV-1 gB has the consensus sequence forsix N-glycosylation sites and contains 10 cysteine resi-dues (Pellett et al., 1985b). These are conserved in gBhomologs in the genomes of HSV-2 (Stuve et al.,1987), Epstein-Barr virus (Pellett et al., 1 985a), cyto-megalovirus (Cranage et al., 1986), and varicella zostervirus (Davidson and Scott, 1986) .

HSV-1 gB is a multifunctional protein that facilitatesthe penetration of virions into cells (Little et al., 1981 ;Sarmiento et al., 1 979a) . Marker rescue experimentswith the mutant HFEMtsBS, a temperature-sensitive

0042-6822/91 $3.00copyright t 1991 by Academic Press, Inc .Al rghts of reproduction n any form reserved .

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QADRI ET AL .

mutant that does not penetrate cells, showed that themutation maps in the extracellular domain of gB (Pellettet al., 1 985b) . In contrast, the syn locus, which pro-motes the fusion of infected and uninfected cells,maps in the intracellular carboxy terminus of the glyco-protein . Mutations in gB that affect the rate of virionentry and the syn phenotype in tsB5 result from singleamino acid changes in the nucleotide sequence of thegene (Bziketat, 1984a). Studies on the assembly of gBshowed that the wild-type glycoprotein forms homo-dimers, and that dimers formed by tsB5 are defective atthe nonpermissive temperature (Claesson-Welsh andSpear, 1986 ; Haffey and Spear, 1980 ; Sarmiento etal.,1979b) and differ in surface configuration from wild-type gB (Chapsal and Pereira, 1988) .

We have characterized a panel of 47 monoclonal an-tibodies to HSV-1 gB (Chapsal and Pereira, 1988 ; Pe-reira at al., 1 982a ; Pereira at al., 1981 ; Pereira et al.,1 982b) and used them in several studies to identifyfunctional domains on the molecule (Kousoulas at at,1984, 1988, 1989 ; Pellett et at, 1985b ; Pereira et at,1989). Nineteen antibodies recognize continuous epi-topes composed of adjacent amino acids ; 28 antibod-ies react with discontinuous epitopes assembled byjuxtaposing amino acids from different regions of themolecule (Chapsal and Pereira, 1988) . Nineteen of theantibodies to discontinuous epitopes have neutralizingactivity, indicating that these functional regions arehighly dependent on tertiary structure .

By nucleotide sequence analysis of HSV-1 gB mu-tants selected for resistance to several potent neutraliz-ing antibodies, we showed that the amino acidchanges clustered in two distinct domains : anHSV-1 -specific domain of continuous residues thatmaps in the amino terminus, and a type-common do-main of discontinuous residues located betweenamino acids 273 and 298 (Kousoulas et al ., 1984,1988 ; Pellett et at, 1 985b) . Subsequently, we showedthat amino-terminal, monomeric derivatives of gB thatlacked the transmembrane region and carboxy ter-minus of the molecule contained all of these continu-ous neutralizing epitopes (Pereira at al., 1989) . In addi-tion, a subset of the discontinuous epitopes was as-sembled on derivatives at least 457 amino acids inlength . Taking these results together with the map lo-cation of amino acid changes in the nucleotide se-quence of antibody-resistant mutants in gB, we con-cluded that a large number of potent neutralizing anti-bodies recognized residues that clustered in a majordomain of discontinuous epitopes assembled from jux-taposing residues between positions 273 and 457 onmonomers of the glycoprotein .

In the present study, we constructed deletion mu-tants, insertion mutants, and point mutants in HSV-1

gB to locate the amino acids which affect the assemblyof discontinuous epitopes . Transient expression of thegB mutants in transfected COS-1 cells showed thattheir pattern of distribution and transport to the cellsurface differed dramatically from that of the wild-typemolecule . All antibodies in the panel reacted with anamino-terminal derivative 690 residues in length, but asubset failed to recognize derivatives 600 residues orshorter. The finding that the longer derivatives formeddimers indicated that residues mapping between resi-dues 600 and 690 affect the conformation of epitopesassembled from discontinuous amino acids juxta-posed on the dimeric form of gB . Insertion of linkers atresidues 391 and 413 caused the loss of certain con-formation-dependent epitopes in this region . A linkerinserted at residue 479 in full-length gB affected theconformation of assembled epitopes in both proximaland distal domains . This mutant glycoprotein wasaberrantly processed and was not translocated to thecell surface. Site-directed oligonucleotide mutagene-sis of cysteines at positions 334 and 382 perturbeddiscontinuous epitopes in the major neutralizing regionproximal to these sites, and distal epitopes formed byassembled amino acids in other regions of the ectodo-main .

MATERIALS AND METHODS

Reagents

Restriction endonucleases, mung bean nuclease,T4 DNA ligase, T4 polynucleotide kinase, and syn-thetic linkers were obtained from New England Biolabs(Beverly, MA) . DNA polymerase I large fragment(Klenow fragment) and calf intestinal alkaline phospha-tase were purchased from Boehringer-Mannheim Bio-chemicals (Indianapolis, IN) and were used accordingto the supplier's instructions . Published procedureswere used forthe construction and analysis of recombi-nant plasmids (Maniatis et al ., 1982) . Synthetic 19-meroligonucleotides used for site-directed mutagenesiswere purchased from the Biomolecular ResourceCenter, University of California San Francisco .

Strains and vectors

Escherichia colt strain DH5 a was used for isolatingand amplifying plasmid DNA, and strain TG2 was usedfor propagating single-stranded bacteriophage M13 .71/18 mutL : :Tnl0 was used for selecting oligonucleo-tide-directed point mutants. E. colt strains were grownin 2X YT medium . For antibiotic selection the mediumwas supplemented with ampicillin (100 µg/ml) and tet-racycline (15 µg/ml) . Plasmid DNA was purified by equi-

librium sedimentation in CsCI according to publishedprocedures (Birnboim et al., 1979) .

Plasmids

Plasmid pRB2080, which contains the EcoRl/Sailsubfragment of BamHl fragment G of HSV-1(F) (Kou-soulas et al., 1984), was used to construct all deletionand insertion mutants in the intact gB gene . PlasmidpRB2002, which contains the Pstl fragment encodingthe 475 amino-terminal residues of gB, was used forinsertion mutagenesis of the major discontinuous-epi-tope neutralizing region of the molecule (Kousoulas etal., 1984). After the insertion of linkers was confirmedwith the appropriate restriction endonucleases, thefragment encoding gB was transferred to the eukary-otic expression vector p91023 (Wong et al ., 1985) orthe derivative vector pMT2 (Genetics Institute, BostonMA) containing the adenovirus major late promoter,the SV40 origin, and, respectively, the tetracycline andampicillin resistance genes . Unphosphorylated linkerswere used for in-frame insertion mutagenesis to avoidmultiple insertion of linkers . Cysteine residues at 334and 382 were replaced with serines by site-directedmutagenesis of pRB2080 cloned in the single-strandedbacteriophage M13, mpl9 (Zoller et al ., 1982) .

The synthetic 1 9-mer oligonucleotide primers weredesigned to change a cysteine anticodon (TGC) to aserine (TCC). (5'-GTCATGGT GGAGACCGACG-3') wasused to change cysteine 334 and the oligonucleotide(5'-TFGCCGATGGAGTCCCCA-3') to change cysteine382 . The anticodon is underlined and the G in eachcase was the mismatch . Potential mutants werescreened with a 32 P-labeled mutagenic primer, and thenucleotide sequence of the clones was determined bythe dideoxy chain-termination procedure (Sanger et al.,1977) . Replicative DNA was purified from the positiveclones, and the blunt-ended BamHl/Sail fragment wascloned into the blunt-ended EcoRl site of the vectorp91023 .

Cells and medium

COS-1 cells were obtained from the American TypeCulture Collection and were grown in Dulbecco's modi-fied Eagle's minimum essential medium, supple-mented with 10% fetal bovine serum .

Monoclonal antibodies

The properties of the panel of monoclonal antibodiesto HSV-1 gB produced in this laboratory and used inthis study were reported (Chapsal and Pereira, 1988 ;Kousoulas et al., 1984, 1988, 1989 ; Pereira et al .,1989 ; Pereira et al., 1981, Pereira et al., 1 982b) .

EFFECT OF MUTATIONS IN 1-15V-1 gB

DNA transfection

Procedures used for the preparation of COS-1 cellsand transfection of plasmid DNAs, using DEAF dex-tran, were reported (Pereira et at, 1989) . Transfectedcells were incubated with DNA for 3 hr then with 10mM n-butyric acid for 12 hr ; after the butyric acid wasremoved, the cells were incubated for a final 24 hr .

Immunofluorescence assays

Procedures for immunofluorescence assays ontransfected cells fixed with acetone were describedpreviously (Pereira etal., 1989). Intact transfected cellswere examined by immunofluorescence assays tomonitor the transport of gB mutants to the cell surface .Forty-four hours after transfection, COS-1 cells on mul-tichambered slides were cooled on ice and washedtwice with ice-cold sterile phosphate-buffered saline(PBS, pH 7.4) . A pool of monoclonal antibodies (1 :100in PBS) reactive with the continuous-epitope domainsD 1 a, D 1 b, and D 1 c was added to the cells and allowedto react for 30 min (Pereira et al., 1989) . Cells werewashed twice with PBS, and FITC anti-mouse conju-gate (1 :100 in PBS; Antibodies, Inc., Davis, CA) wasadded . After 30 min, the cells were washed twice withPBS, air-dried, and fixed with 0 .1% paraformaldehydefor 15 min at room temperature. They were thenwashed with distilled water, mounted, and observed inan Olympus epifluorescence microscope .

Radiolabeling and immunoprecipitation

Transfected COS-1 cells were radiolabeled with[ 35S]methionine (50 pCi/ml ; DuPont-New England Nu-clear, Inc .) for 4 hr at 44 hr after transfection . Immuno-precipitation assays were done with culture mediumand with transfected cells extracted with 1 % Nonidet-P40 and 1 % sodium deoxycholate . Samples were elec-trophoresed in denaturing polyacrylamide gelscontaining sodium dodecyl sulfate, as describedpreviously (Pereira et at, 1980) . To determine whetherdimers were formed, the samples were electropho-resed in polyacrylamide gels under native conditions,using buffers containing 0 .1% SDS, without heat or3-mercaptoethanol . They were then transferred to ni-trocellulose, and reacted with monoclonal antibodiesas previously described (Chapsal and Pereira, 1988 ;Cohen et al., 1 986) .

RESULTS

Construction of mutants in the HSV-1 gB gene

To identify the amino acids that affect the assemblyof discontinuous epitopes on monomers and dimers of

1 3 7

138

QADRI ET AL .

A.

HSV-1 gB 1

pN3393

pPS600

pTS690

pTRO690

C.

pNB391

pBB413

pS8479

pPSa600

pNhB810

pNha4ep810

D .

pCys334

pCys382

B .

NH2

j

V"' 3

Bglll

Y475391

Bglll

V 475413

Bglll

Y

ser4cye 382

FIG. 1 . Schematic representation of HSV-1 gB and mutants . The structure of the wild-type gB molecule of 874 amino acids is shown in A(Pellett et at., 1985b). The hydrophobic transmembrane region is depicted ass hatched box . Stick models of the mutant constructs in gB areasfollows : deletion mutants (B), linker-insertion mutants (C), and site-directed mutations in cysteine residues (D) . The last residue of the amino-ter-minal derivatives is indicated by a number. The synthetic oligonucleotide linkers and their insertion sites in gB are shown by an inverted triangle .a4ep refers to the H1091 epitope of the HSV-1 a4 protein (Hubenthal-Voss et al ., 1988) . Plasmids are indicated to the left of each construct .Names used to refer to the mutant glycoproteins are indicated to the right .

HSV-1 gB, we constructed three sets of mutants . Thefirst set contained deletions in the carboxy terminusand transmembrane regions of the molecule . The sec-ond set had linker-insertions in the ectodomain andintracellular regions of the molecule . The third set con-tained site-specific changes in cysteine residues lo-cated in the major discontinuous-epitope neutralizingdomain . Stick figures illustrating wild-type gB (Fig . 1A)and the mutations in the gene (Fig . 1 B-1 D) are shown .Sequences of amino acids altered by the mutations areshown in Fig . 2. A description of each gB mutant fol-lows .Deletion mutants . The set of deletion mutants con-

tained four constructs with deletions of varying lengthsfrom the carboxy terminus of gB (Fig . 1 B) . PlasmidspNS393 and pPS600 contained an Spel linker, whichencodes termination codons in all three readingframes, inserted at the Ncol and the Pvul restriction

9B .(1.393)

spel

7.Spel

7690

9B-(1690)

EcORI

Y-674

gB-(1690A849)690

849

S[a7ll

7600

Bglll

=7 67481Da4ep

610ser

T0ys330

COIOH

®'874

Wild-type 9B

®674

g B-(1 .600)

gB-(1 .475-Lk391)

gB{1 .475-Lk413)

674

gB-(Lk479)

gB .(Lk600)

gB-(Lk810)

g8-(a4ep810)

874

gB-Cye-5

®874

;;B-Cye6

sites, respectively . Both mutants, designated as gB-(1-393) and gB-(1-600), respectively, lacked the trans-membrane region and a portion of the ectodomain andcarboxy terminus of the glycoprotein . PlasmidpTRA690 contained a deletion of a Tth1111 fragmentand an in-frame insertion of an EcoRl linker . The result-ing derivative lacked 160 amino acids of gB mappingbetween residues 690 and 849 and encoded residuesThr-Glu-Phe-Leu at the fusion site followed by the car-boxy-terminal 25 amino acids of gB . Plasmid pTS690was a derivative of pTRA690 that contained an Spellinker encoding termination codons at the EcoRl site .Mutants encoded by pTS690 and pTRS690, desig-nated as gB-(1-690) and gB-(1-690A849), containedthe entire ectodomain of gB .

Insertion mutants . The second type of constructswas insertion mutants (Fig . 1C) . For these mutants, theamino acid sequence and the charged residues in the

Mutant

gB-(1-475-1-1,391)

gB-(1 . 475-L1,413)

gB-(Lk479)

g13 (11,600)

gB-(LkB10)

g1-(u4ep-810)

region of the insertions is shown in Fig . 2. ConstructpNB391 contained an in-frame insertion of a Bglll linkerat the Ball restriction site of plasmid pRB2002, whichcontains the Pstl subfragment encoding the amino-ter-minal 475 residues of gB . The mutant glycoprotein wasdesignated as gB-(1-475-Lk391) . Construct pBB413contained an in-frame insertion of a Bglll linker at theNcol restriction site of pRB2002, and the mutant wasdesignated gB-(1-475-Lk413) . This group of mutantsalso included three clones with linkers inserted in-frame in the intact gB molecule at residues 479, 600,and 810. Plasmid pSB479 contained a BgIll linker atresidue 479, and the mutant was designated as gB-(Lk479). pPSa600 contained a Sall linker at residue600, and the mutant was named gB-(Lk600) . PlasmidpNhB810 contained a Bglll linker at residue 810 of theintracellular domain of gB, and the mutant was desig-nated as gB-(Lk810) . A second insertion mutant,pNha4ep, was constructed in the carboxy terminus atresidue 810 by inserting a synthetic oligonucleotideencoding a heterologous HSV-1 a4 epitope, H1091(Hubenthal-Voss et al., 1988) . The mutant was desig-nated as gB-(a4ep-810), and the sequence of the 17-amino-acid insertion is shown in Fig . 2 .

EFFECT OF MUTATIONS IN HSV-1 qB

Amino Acid Sequence

Gin lie Cys Met

Val Asp Glu Asp Lou Ala Arg Asp Ala Mel

Asp Arg lie Phe Ala Arg Arg Tyr Asn Ala391

392

Gly Lys lie Phe Pro

lie Lys Val Gly Glu Pro Gin Tyr Tyr Lou

Asn Gq Gly Phe Leu lie Ala Tyr Gin Pro412

414

Glu Asp Leu Pro

Ser lie Glu Phe Ala Arg Lou Gin Phe Thr

T Asn His lie Gin Arg His Val Asn Asp479

489

.Gly Arg Pro

+

+ -

+ +Asn Asn Glu Leu Arg Leu Thr Arg Asp Ala

Glu Pro Cys Thr Val Gly His Arg Arg Tyr599

601

Ala Asp Lou Lou

Gly Gly Asp Pre Asp Glu Ala Lys Lou

Ala Glu Ala Arg Gin Met lie Arg Tyr Met810

811

Ala Ala Gly Glu Asp Ala Gly Asp Asp Val Ser Pro Arg Glu Lou Ala Len

Gly Gly Asp Phe Asp Glu Ala Lys Lou Ala Glu Ala Arg Glu Met lie Arg Tyr Metaip

all

139

FIG . 2 . Amino acid sequence at the site of the linker-insertion in HSV-1 gB . The amino acid sequences flanking the site of the insertion areindicated and the sequence of inserted residues is shown above each mutant . Numbers below the sequence refer to the amino acids at the siteof the insertion . Al,,, was deleted from mutant gB-(1-475-Lk413) and Ile 000 from mutant gB-(Lk600). Charged residues in the sequence areindicated as large polar (t, ) and small polar (+, -) .

Site-specific mutants. The third set of constructswere point mutants in which serine residues were sub-stituted for cysteines by oligonucleotide-directed mu-tagenesis (Fig . 1 D). Plasmids pCys334 and pCys382encode glycoproteins that contained point-mutationsat cysteines 334 and 382 in full-length gB and weredesignated as gB-Cys-5 and gB-Cys-6, respectively .The synthetic oligonucleotides used to direct theamino acid changes were described under Materialsand Methods .

Intracellular location of gB mutants

The first series of experiments was designed tocompare the intracellular location of various mutantforms of gB with that of the wild-type molecule by im-munofluorescence reactions on transfected cells .Cells were permeabilized 48 hr after transfection andreacted with a pool of monoclonal antibodies to contin-uous-epitope domains Dl a, D1 b, and D1 c, which mapin the amino terminus of gB (Table 1) . We reasonedthat epitopes mapping in these domains would be re-tained on derivatives mutagenized in distal sites. Re-

140

suits of these experiments are shown in Fig . 3 (A-L)and are summarized as follows .

Wild-type gB displayed a strong perinuciear, globularstaining pattern indicative of Golgi localization and nor-mal processing through the exocytic pathway (A) . Ofthe deletion mutants, the amino-terminal derivative gB-(1-393) showed a reticular staining pattern in the cyto-plasm and strong staining of the nuclear membrane(B) . The longer derivatives gB-(1-600) (D), which lackeda small portion of the ectodomain as well as the entiretransmembrane and carboxy domains, and gB-(1-690)(E), which lacked just the transmembrane and intracel-lular regions, assumed a reticular diffuse appearancethat indicated aberrant processing . Similar findingswere noted with gB-(1-690A849), which retained asmall portion of the carboxy tail (F) . With regard to the

QADRI ET AL .

FIG . 3 . Internal immunofluorescence photographs of COs-1 cells transfected with wild-type HSV-1 gB and mutants . Cells transfected with gBconstructs were reacted with a pool of monoclonal antibodies to continuous-epitope domains D1 a, D1 b, and Ms . Wild-type gB (A), gB-(1-393)(B), gB-(1-475-Lk391) (C), gB-(1-600) (D), gB-(1-690) (E), gB-(1-6904849) (F), gB-(Lk479) (3), gB-(Lk600) (H), gB-(Lk810) (I), gB-(a4ep8l0) (J),gS-Cys-5 (K), gB-Cys-6 (L) . X600 .

linker-insertion mutants, gB-(1-475-Lk391) (C) showeda diffuse cytoplasmic staining pattern, indicating thatthe mutant was located in the endoplasmic reticulum .Similar results were found with gB-(1-475-Lk413) (notshown). These results agreed with results of ourprevious analysis of truncated gB derivatives thatlacked the carboxy terminus (Pereira et al., 1989). In-sertion mutants in the extracellulardomain of the intactglycoprotein, gB-(Lk479) (G) and gB-(Lk600) (H), and inthe intracellular domain, gB-(Lk810) (1) and gB-(a4ep-810) (J), were distributed throughout the cytoplasm in apattern different from that of wild-type gB . As to theglycoproteins containing point mutations at cysteineresidues, gB-Cys-5 (K) and gB-Cys-6 (L), their cytoplas-mic distribution was generally punctate, but with somepatchy areas that suggested local aggregation in cer-

tain regions of the cell . This pattern differed signifi-cantly from that found with wild-type gB and with theother mutants .

Cell-surface transport of gB mutants

Studies on transport of glycoproteins specified byvesicular stomatitis and influenza viruses indicate thatthe native conformation of the glycoprotein is requiredfor transport to the cell surface, and that certain muta-tions in the extracellular domain disturb the processingof these glycoproteins (Gething et al., 1986 ; Kreis andLodish, 1986 ; Rose and Bergmann, 1982) . To deter-mine whether the mutant forms of gB were transportedand stably associated with the cell surface membrane,we performed surface immunofluorescence assays oncells transfected with the various gB constructs . Thetransfected cells were reacted with the pool of mono-clonal antibodies to the Dl domains prior to fixation, asdescribed under Materials and Methods . Results ofthese experiments are shown in Fig . 4 (A-I) .

Wild-type gB was transported in large amounts tothe cell surface (A) . In contrast, only trace amounts ofthe truncated glycoproteins specified by the deletionmutants gB-(1-393) (not shown), gB-(1-600) (B) and gB-(1-690) (C) were stably associated with the cell surfacemembrane. Although gB-(1-690z%849) retained only 25carboxy-terminal residues of the intracellular domain,these appeared to have some effect in stabilizing thismutant in the cell-surface membrane (D) . Analysis ofthe linker-insertion mutants in intact gB showed thatthe mutant gB-(Lk600) (E) was associated with the sur-face membrane. In contrast, no trace of the insertionmutant gB-(Lk479) could be detected on the surfacemembrane (data not shown) . Mutants gB-(Lk810) andgB-(«4ep-810), which contained insertions in the intra-cellular carboxy terminus, were transported to the cellsurface (H and I) in amounts comparable to those ofwild-type gB . Surface immunofluorescence analysis ofthe cysteine mutants gB-Cys-5 (F) and gB-Cys-6 (G)showed that both reached the cell surface .

Epitopes expressed by deletion mutants in gB

We previously located the amino acids that contrib-ute to the formation of epitopes recognized by 22 of 47monoclonal antibodies to HSV-1 gB by constructing aset of amino-terminal derivatives that lacked the trans-membrane region and carboxy terminus of the mole-cule (Pereira et al., 1989). The derivative 475 aminoacids in length failed to form dimers but did express asubset of discontinuous epitopes . Based on these re-sults, it was reasonable to expect longer amino-termi-nal derivatives of gB to express the epitopes of theremaining antibodies in the panel . We therefore de-

EFFECT OF MUTATIONS IN HSV-1 9B 141

signed experiments to assess the reactivity of theseantibodies on the constructs that expressed mutantsgB-(1-600), gB-(1-690), and gB-(1-690A849), contain-ing most or all of the ectodomain of the glycoprotein(Fig . 1). These were compared in immunofluorescencetests with wild-type gB and with the amino-terminalderivative gB-(1-393) for expression of various epi-topes . Results of these experiments are summarized inTables 1 and 2 . The salient features of the results areas follows .

Derivative gB-(1-393) expressed continuous-epitopedomains D1 a, D1 b, Dtc, and D2c but failed to expressthe discontinuous-epitope domains D2a and D2b .These results agreed with our previous analysis of de-rivatives approximately the same length . In contrast,the longer mutant gB-(1-600) expressed the discon-tinuous-epitope domains D2a, D2b, D3a, and D3b aswell as continuous-epitope domains Dl a, D1b, D1c,and D2c. We located one new antigenic domain in thisregion between residues 475 and 600, and designatedit as Ddb . It contains five discontinuous butnonneutraiizing epitopes . It should be mentioned thatcertain antibodies failed to recognize g B-(1-600) . Deriv-atives gB-(1-690) and gB-(1-6900849), which con-tained the entire ectodomain (the hydrophobic trans-membrane sequence begins with amino acid 697), ex-pressed all of the epitopes in the panel . We locatedfour novel domains between amino acids 600 and 690 :Dd5a and DdUb, which contain three and nine discon-tinuous epitopes, respectively ; and Dc4a and Dc4b,which contain five and three continuous epitopes, re-spectively. The three epitopes in Ddba bind neutraliz-ing antibodies .

Next, extracts of cells transfected with selected mu-tant constructs were immunoprecipitated with antibod-ies whose epitopes require the participation of aminoacids located between positions 600 and 690 . Pools ofantibodies to the D1 domains were used as positivecontrols . Figure 5 shows the electrophoretic profiles ofimmune precipitates after reactions of gB(1-690) (lanes1-4) and gB(1-600) (lanes 11-14) with pools of mono-clonal antibodies representing different antigenic do-mains . Reactions with selected antibodies to discon-tinuous epitopes in domains DdSa (H420, H 1373, andH1695), DdSb(H121), D2b (H1828), and D3b(H1693)are also shown (lanes 5-10 and 15-20) . These resultsconfirmed the presence of epitopes that were deter-mined by immunofluorescence tests, with two excep-tions. First, a pool of antibodies to Dc4a reacted poorlyin immunoprecipitation tests with both mutants . Sincea weak signal was found in reactions of the Dc4a anti-body pool with 9B-(l -600), it is likely that some epi-topes include residues overlapping the terminus ofthis mutant. Second, antibodies H1828 and H1693

142

reacted weakly with gB-(1-600), whereas they had notrecognized this mutant in immunofluorescence tests .The occasional difference observed may result from asmall change in the conformation of the epitope, per-haps by folding of the molecule induced by extractingthe glycoproteins in nonionic detergents for immune-precipitation reactions .

Residues required for dimerization of gB

Characterization of the panel of 47 monoclonal anti-bodies to HSV-1 gB showed that 28 antibodies reactwith discontinuous epitopes dependent on the confor-mation of assembled domains (Table 1) (Chapsal andPereira, 1988) . Analysis of the antigenic structure of gBmade at the nonpermissive temperature by tsB5, a mu-

OADRI ET AL .

Fio . 4 . Surface immunofluorescence photographs of COS-1 cells transfected with wild type HSV-1 gB and mutants and stained with a pool ofmonoclonal antibodies to continuous-epitope domains D1 a, D1 b, and D1 c . Wild-type gB (A), gB-(1-600) (B), gB-(1-690) (C), gB-(i-6906849) (D),gB-(Lk600) (E), gB-Cys-5 (F), gB-Cys-6 (G), gB-(Lk810) (H), g3-(e4ep810) (I) .

tant that produces unstable dimers, revealed that asubset of discontinuous epitopes present on wild-typedimers was lost on the mutants. We later found that 11of these discontinuous epitopes were formed onamino-terminal derivatives containing 457 residuesand lacking a portion of the ectodomain, the trans-membrane region, and the carboxy terminus (Pereira etal., 1989) . The finding that the truncated mutants failedto form dimers indicated that these discontinuous epi-topes were formed by juxtaposing residues on gBmonomers .

To locate the amino acids participating in the forma-tion of gB dimers and to determine whether the discon-tinuous epitopes on the longer derivatives were depen-dent on the dimeric configuration, COS-1 cells trans-fected with the mutant constructs were extracted with

EFFECT OF MUTATIONS IN HSV-1 gB

143

TABLE 1

GROUPING OF CONTINUOUS AND DISCONTINUOUS EPITOPES INTO DOMAINS ON HSV-1 gB

Note . References cited :Chapsal and Pereira, 1988 .

° Follett et al ., 1985b .° Kousculas et al., 1988 ."Kousoulas et al., 1989 .e Pereira et al., 1989 .r Present study .

Type/location' Domain

Monoclonal

Cellantibody

surfaceSerotype Neutralizereactive

virus Domain/epitope mapping Ref

Contin Dia H1817

I TO

,- Maps in peptide 1-20 c-eH1830

+ TS Maps in peptide 1-20H1839

+ TS

+ Maps in peptide 1-20D1b H1392

+ TS

+ Mutation at Ala ., c-eH1396

+ TS

+ Maps on peptides 16-35, 35-50H1397

+ TS

+ Mutation at Thr„D1c H1838

+ TO

+ Overlaps residues 1-47 c, eD2c H1781

+ TO

+ Maps between 440 and 457 e

D3c H1359

+ TCDomain lost in gB-(1-475-Lk391)Maps between 457 and 475 eDomain lost in gB-Cys-5, gB-Cys-6, gB-(1-475-Lk391),

gB-(1-475-Lk413), and gB-(1-600)H1385 TOH1393 TS

Dc4a H1394 TO Domain maps between 600 and 690, partially

H1411 TOoverlapping gB-(1-475), lost in gB-(1-690,X849)

H1399 TO

- Epitope lost in Cys-5H1382 TS

-H1757 TS

-Dc4b H1316

+ TO Domain maps between 600 and 690, lost on Cys-6 fand gB-(1-690,X849)

H1163

+ TOH336

+ TODiscontin/ D2a H233

+ TO

+ Domain not present on gB-(1-393), lost on gB-(1-475- b, c, emonomer

H126

+ TO

+

Lk391), gB-Cys-6Mutation at His 208/assembled 1-457Mutation at Asn, 73 /assembled 1-457

H1375

+ TO

+ Mutation at Gln z„/assembled 1 457

H1435

+ TOEpitope lost in gB-(Lk479)Mutation at Tyr27A /assembled 1 . 457

D2b H1815

+ TO Domain not present on gB-(1-393), assembled ebetween 1 and 457, lost on gB-(1-475-Lk391) andgB-(1-475-Lk413)

H1819

+ TO

+Epitope lost in gB-(Lk479), gB-Cys-6

H1828

+ TO

- Epitope lost in gB-(1-600)D3a H352

+ TO

+ Domain assembled on gB-(1-475), lost on g8-(1-475- e

Dab H1693 TO

Lk391), gB-(1-475-Lk413), gB-Cys-5, gB-Cys-6Epitope lost in gB-(Lk479)Domain assembled on gB-(1-475) but lost in gB-(1- e475-Lk391), gB-(1-475-Lk413), and gB-(1-600)

H1708

+ TOH1376

+ TO Epitope lost in gB-(1-600)Discontin/ DdSa H420

+ TO

+ Domain maps between 600 and 690Dinner

H1373

+ TOEpitope lost in g9-Cys-6Epitope lost in gB-Cys-5

H1695

+ TO Lost in Cys-5 and Cys-6DdSb H121

+ TO Domain maps between 600 and 690H1457

+ TOH157

+ TO

- Epitope lost in gB-(Lk479)H189

+ TO

-H1711 TOH1727

+ TO Epitope lost in gB-Cys-6H1807

+ TOH1814

+ TOH1823

+ TODd6 H1783

+ TC

- Domain maps between 475 and 600 and weak on

H1798

+ TO

gB-(Lk479)Epitope lost in gB-Cys-5

H309

+ TOH120

+ TOH146

+ TO Epitope lost in gB-Cys-5

Note. D 1 rotors to (D 1 a, D 1 b, D 1 e) .' Weakly reactive .

nonionic detergents, electrophoresed in native poly-acrylamide gels in native or denaturing sample buffer,transferred to nitrocellulose, and reacted with a pool ofantibodies to domains D1 a, b, and c as describedunder Materials and Methods . Results of these experi-ments are shown in Fig . 6 .

Wild-type gB formed dimers (D) that dissociated inthe presence of /3-mercaptoethanol and heat (M), asdid the linker-insertion mutants gB-(Lk600) and gB-(a4ep810) . Interestingly, mutant gB-(Lk479), whichfailed to undergo translocation to the cell surface, alsoformed dimers . The longest truncated mutant, gB-(1-690), also formed stable dimers, but gB-(1-600) did not .These results suggest that the first 600 residues of thegB ectodomain do not contain all of the signals re-quired for dimerization of the molecule . Moreover, theyshow that discontinuous epitopes in domain Dd6formed by residues 475 to 600 were assembled on gBmonomers. Mutant gB-(1-690), however, formed threebands in native gels : one band with the electrophoreticproperties of a monomer (M) and two bands that mi-grated more slowly . The band designated as D had thehigh-molecular-weight properties of a dimer thatagreed with the approximate size of the truncated mu-tant . The second band was larger and not well resolved

in native gels, and may contain multimers of the mu-tant. We have found that certain forms of the truncatedgB mutants and other full-length mutants associatewith the glucose-regulated protein, GRP78 (L . Pereira,I . Qadri, D . Navarro, and C . Gimeno, manuscript sub-mitted) . This complex is stable under nondenaturingconditions, which could account for the more slowlymigrating bands in profiles of certain mutants . Bothhigh-molecular-weight forms migrated as monomersafter denaturation, suggesting that they are not aggre-gates . That mutant gB-(1-690) formed dimers indicatedthat discontinuous epitopes in domains DdSa andDd5b, expressed on this derivative but not shorterones, are dimer-dependent. Native forms of the glyco-protein expressed by gB-(1-6907849), which contains25 amino acids of the carboxy tail of gB fused to theectodomain of the molecule, also formed dimers . Aswith gB-(1-690), the monomeric form of this mutantwas present under native conditions ; under denaturingconditions (D) the high-molecular-weight bands mi-grated as monomers (M) . It should be mentioned thatthe findings obtained with the native gel system wereconfirmed by analysis of the gB mutants on sucrosegradients (D . Navarro, I . Qadri, and L . Pereira, manu-script in preparation) .

144 QADRI ET AL .

TABLE 2

ANTIGENIC PROPERTIES OF MUTANTS IN HSV-1 gB

Domains fully retained Domains fully lost Partial domains (opitopes lost)

Plasmid Mutant protein Continuous Discontinuous Continuous

Discontinuous Continuous Discontinuous

pNS393 gB-(I-393) Dl, D2c none none

D2a, D2b none nonepPS600 gB-(1-600) DI, Dec, Dc4a' D2a D2b, D3a, Dd6' D3c

none none D3b(H1693,H1376)pTR690 gB-(1-690) DI, D2c, D3c, D2a .. D2b, D3a, Dab, none

none none none

pTRA690 gB-(1-690A849(Dc4a a Dc4b

DI, D2cDd6, DdSa, Dd5b

D2a, D2b, D3a, Dab, none

none none none

pNB391 gB-(1-475-Lk391) DlDd6, oars, Dd5b

none D2c, D3c

D2a, D2b, none none

pBB413 gB-(1-475-Lk413) Dl, D2c D2a (H233e)D3a, D3b

D3c

D2b, D3a, D3b none nonepSB479 gR-(L.k479) DI, D2e, D3c, D3b, Ddba, Dd6' none

none none D2a(H1375)Do4a, Dc46 Dd5b (H I783') D2b (H1815)

D3a (H352)

pPSa600

pNhB810

pNha4ep

pCys334

gB-(Lk600)

gB-(Lk810)

gB-(n4ep-810)

gB-Cys-b

D1, D2c, D3c,Dc4a,Dc4b

D1, D2c, D3c,Dc4a,Dc4b

DI, D2c, D3c,Dc4a,Dc4b

DI, D2c, Dc4b

D2a, D2b, D3a, D3b,Dd6, DdSa, Dd5b

D2a, D2b, D3a, D3b,Dd6, Dd5a, DdSb

D2a, D2b, D3a, D3b,Dd6,Dd5a,Dd5b

D3b, Dd5b

none

none

none

none

none

none

none

D3a

none

none

none

D3c(H1359, H1385)

Ddbb(H157)none

none

none

DdEa(HI373,H1695)Dc4a (111399) Dd6(H146,1-1783)

pCys382 gB-Cys-6 D1, D2c, Dc4a D3b, Dd6 D3c, Dc4b

D3a none D2a (H233, 11126, Hi 375)D2b(H1815)DdSa (H420, H 1695)Dd5b (01727)

gB41.690)

EFFECT OF MUTATIONS IN HSV-1 gB

145

pools

antibodies

pools

antibodies

FIG . 5. Immunoprecipitation patterns of gB-(1-600) and gB-(1-690) with pools of monoclonal antibodies to different antigenic domains and withindividual monoclonal antibodies . The precipitated mutant glycoproteins are indicated : solid arrow, gB-(1-690) ; dashed arrow, gB-(1-600) .

Results of these experiments indicated that the residues 600 and 690 in the ectodomain of the mole-amino acids contributing to the assembly, conforma- cule and are independent of the hydrophobic trans-tion, and stability of gB dimers are located between

membrane sequence and of the intracellular carboxyterminus .

t

-M

FIG . 6. Immunoblot analysis of HSV-1 gB monomers and dimers .Cell lysates transfected with wild-type and mutant gB constructswere electrophoresed in 7 .596 polyacrylamide gels under native con-ditions, transferred to nitrocellulose, and reacted with a pool ofmonoclonal antibodies to continuous-epitope domains Dta, D1b .and D1 c . Labels above lanes indicate wild-type gB and mutant con-structs ; N, native sample buffer (no i3-mercaptoethanol or heat) ; D,denaturing sample (buffer contained 2 0/b f3-mereaptoethanol andsamples were boiled 5 min prior to electrophoresis) . Arrows to theright point to gB dimers (D) and monomers (M) . o beside band indi-cates monomeric form of truncated mutant . gB-t1-x690) designatesconstruct gB-(1-690zX849) .

gBf1.600)

Effect of directed mutations on the assemblyof discontinuous epitopes on gB

Results of our previous studies (Kousoulas et a/.,1988 ; Pellett et al., 1985b ; Pereira et al., 1989) indi-cated that some of the conformation-dependent do-mains of gB are assembled from discontinuous aminoacids on monomeric forms of gB . It is highly likely thatcysteine residues forming intramolecular disulfidebonds play a significant role in the assembly of discon-tinuous epitopes by juxtaposing distal amino acids onthe molecule .

In the next series of experiments, we used the mu-tants with linker insertions and point mutations in cys-teine residues to determine the effect of thesechanges on the surface configuration of the molecule .Changes in conformation were monitored by alteredreactivity of specific antibodies . Epitopes expressed bythe mutants in immunofluorescence tests with thepanel of monoclonal antibodies are listed in Table 2 .Results of these studies are summarized as follows .

Insertion mutants in the amino-terminal derivativegB-(1-475) . The linker-insertion mutants in the amino-terminal derivative of gB, gB-(1-475-Lk391) and gB-(1-475-Lk413), retained all of the continuous epitopesmapping in domains D1a, D1b, and D1c. gB-(1-475-Lk413) also expressed domain D2c and the discon-

s m0m 0 m ma so md fD Y_J

ur rD

JD.

mm

D N D N

mm

D N

mm

D N 0

-C

N D N D N

1 46

QADRI FT AL.

sinuous domain D2a but lost D2b, whereas gB-(1-475-Lk391) did not express either D2a or D2b . Both mu-tants fully lost the discontinuous-epitope domainsD3a and D3b as well as the continuous-epitope do-main D3c .

Insertion mutants in intact gB. Of the linker-insertionmutants in the ectodomain of intact gB, gB-(Lk479) un-derwent the most antigenic change, losing discon-tinuous epitopes in four domains, D2a, D2b, D3a, andDdSb. As noted earlier, the processing of gB-(Lk479)was extremely aberrant, as evidenced by the intracel-lular and cell-surface patterns of immunofluorescence .Detailed analysis of the processing and transport ofthis mutant has revealed significant processing de-fects relative to insertion mutants in other regions of gB(D . Navarro, I . Qadri, and L . Pereira, manuscript in prep-aration). In contrast to the findings with gB-(Lk479),gB-(Lk600) retained all of the epitopes specified by thewild-type molecule . The insertion mutants in the intra-cellular domain, gB-(Lk810) and gB-(a4ep-810), failedto show any detectable changes in the conformation ofdiscontinuous or continuous epitopes, suggesting thatcarboxy-terminal mutations do not affect the configura-tion of the ectodomain .

Point mutants in cysteine residues . The point mutantin cysteine 334, gB-Cys-5, showed detectablechanges in antigenic structure, as reflected bythe reac-tions of monoclonal antibodies . Continuous-epitopedomains Dla, Dlb, Dtc, D2c, and Dc4b were re-tained, as were discontinuous-epitope domains D3band Dd5b, but D3a was lost . Some discontinuous epi-topes in domains Ddb and DdSa proximal to the trans-membrane region were lost . Some continuous epi-topes mapping in domains D3c and Dc4a were alsolost. The point mutant in cysteine 382, gB-Cys-6,showed a different pattern of antigenic changes . Con-tinuous-epitope domains Dla, D1b, D1c, D2c, andDc4a were completely retained, as were discon-tinuous-epitope domains D3b and Ddb . Two continu-ous-epitope domains, D3c and Dc4b, and one discon-tinuous-epitope domain, D3a, were fully lost . Somediscontinuous epitopes in domains D2a, D2b, DdSa,and DdSb were also lost . These results indicated thatgB-Cys-6 had undergone profound antigenic changes .

DISCUSSION

In this study, we generated three types of mutants inHSV-1 gB to locate the amino acids which contributeto the formation of epitopes recognized by a panel ofmonoclonal antibodies, 18 of which had neutralizingactivity . The first type of constructs were deletion mu-tants that lacked portions of the ectodomain, trans-membrane region, and intracellular carboxy terminus .

The second type were insertion mutants constructedby in-frame insertion of linkers and a synthetic oligonu-cleotide into restriction sites in the ectodomain andintracellular carboxy terminus . The third type con-tained site-directed mutations in which cysteine resi-dues mapping in the major discontinuous-epitope neu-tralizing region were changed to serines . New insightsinto the surface configuration of gB, the assembly ofconformation-dependent epitopes on monomers anddimers, and the transport of this glycoprotein are de-scribed below .

Mutations that affect discontinuous epitopes

We previously located the epitopes recognized by 22of 47 monoclonal antibodies to HSV-1 gB by mappingthe amino acid changes that conferred resistance toantibodies with neutralizing activity, and found thatthese epitopes clustered in two regions of the ectodo-main (Kousoulas et al., 1988 ; Pellett et al., 1 985b). Con-tinuous neutralizing epitopes mapped in the aminoterminus between residues 32 and 47 and potentcross-reacting antibodies recognized a domain of dis-continuous epitopes between residues 273 and 298 .Subsequently, by reacting these antibodies withamino-terminal derivatives containing different lengthsof the ectodomain of gB, we determined that additionalresidues were required to assemble the discontinuousepitopes in this domain (Pereira et al., 1989) . This ma-jor neutralizing region was formed on derivatives 457amino acids long, which indicated that a substantialnumber of residues were required for its conformation .In the present study, we confirmed the requirement foradditional residues to promote the folding of assem-bled epitopes by analyzing longer derivatives of gB . Wealso located the amino acids that form the remaining25 epitopes recognized by the panel of antibodies andfound that these residues cluster between positions600 and 690 . In all, 14 antigenic domains have beenidentified, of which 7 are conformation-dependent ; 8bind neutralizing antibodies and cluster in three spa-cially separate regions of gB . A summary of the group-ing of epitopes into these domains, as determined by avariety of techniques in this and our previous studies, isgiven in Table 1 . A schematic representation of thetopology of the antigenic domains on gB is shown inFig. 7 .We have begun to deduce the configuration of dis-

continuous epitopes by analyzing the secondary struc-ture of the insertion sites and point mutations in gB .Figure 8 shows the map location of these mutationsrelative to the predicted secondary structure of thewild-type molecule, which is based on a figure pub-lished by Pellet etat (1985b) . It also shows the location

EFFECT OF MUTATIONS IN HSV-1 gB

147

of the mutations detected by nucleotide sequenceanalysis of mutants resistant to neutralizing antibodies .These locations, two in continuous epitopes (1, 2) andfour in discontinuous epitopes (3, 4, 5, 6), are dis-cussed relative to insertion mutants in different sites onthe gB molecule as follows .

Insertion mutants in the ectodomain of gB . The in-sertion mutant gB-(1-475-Lk391) lost four discon-tinuous domains and mutant gB-(1-475-Lk413) lostthree of these domains (Table 2). Analysis of the sec-ondary structure of the insertion sites in gB indicatedthat a-helix 9 (7) and 0-sheet 4 (8) were perturbed inthese mutants . Loss of domain D2a in gB-(1-475-Lk391) (7) shows that distal amino acids are involved inthe conformation of the discontinuous epitopes be-tween 273 and 298 (3, 4, 5, and 6). Conformationalchanges in this mutant may have arisen from a cys-teine in the linker that led to the improper pairing ofother cysteines into disulfide bonds . Two cysteines,Cys-5 and Cys-6, are located in the intervening se-quences between the insertion sites and the affectedepitopes. Analysis of the insertion site of the linker ingB-(Lk479) indicated that some epitopes in the discon-tinuous-epitope domains are sensitive to perturbationsin /3-sheet 5 (9) . In contrast, the antigenic properties ofthe mutant gB-(Lk600) were not altered . It is of interestthat the insertion site in this mutant (10) does not per-turb any predicted structural conformation . Results ofthese studies have begun to define the boundaries ofepitopes in the major neutralizing region and suggestthat the conformation of this region does not dependon residues adjacent to the transmembrane region .

• 7• 27• 80• 8 4H1823

FIG . 7 . Topographic map of the amino acids located in antigenic and functional domains of HSV-1 gB . Domains on gB are designated by filledellipses on the linear diagram of the glycoprotein . Epitopes formed by residues within each domain are listed in boxes . Amino acid numbers areshown below the diagram . Locations of the transmembrane (TM) domain, entry, and syn mutations are based on nucleotide sequence analysisof the gB gene (Bzik et al., 1984a ; Pellet etal., 1985b). Enclosed boxes indicate the discontinuous epitopes in domains formed on assembled gBhinders .

Point mutants in the ectodomain of gB . Studies onpoint mutations in Cys-5 and Cys-6 indicate that theseresidues play a significant role in maintaining the con-formation of discontinuous epitopes in several do-mains on gB (Fig . 8). gB-Cys-6 lost four neutralizingepitopes in proximal domains D2a and D2b and someepitopes in distal domains DdSa and DdSb (Table 2) .Epitopes in domains DdSa and Dd6 were also lost inthe gB-Cys-5 mutant. These gross perturbations in sec-ondary structure indicate that these cysteines are in-volved in disulfide bonds . Analysis of site-directed mu-tants in HSV-1 glycoprotein D (gD) similarly showedthat altering certain cysteine residues in the extracellu-lar domain causes the loss of discontinuous epitopes(Wilcox et al., 1988) . It was also found that the gD mu-tants were aberrantly processed and contained onlyhigh-mannose-type carbohydrates . The study con-cluded that six of the seven cysteine residues are criti-cal to the correct folding, structure, and processing ofgD and may form three disulfide-bonded pairs . At thepresent time, we are unable to draw conclusions aboutthe pairing of cysteine residues into disulfide bonds onthe gB molecule based on the analysis of two mutants .We are currently mutagenizing each cysteine in gB togenerate a set of point mutants for analysis of the con-tribution of cysteine residues to the conformation ofassembled epitopes .

Structural features and functional domains of gB .Certain features of the surface configuration and prop-erties of the antigenic domains on gB merit discussion .Of particular interest are the location and the nature ofthe potent neutralizing regions on gB . Surface immune-

nt SynTM

NH2 i== illllllllllllu COOH874

20 4

0

28 7 4 5

I dependentDiner-

D1a D1b D1c D2a D2b D2c D3a D3b D3c Dd6 Dc4a Dd5aH1817 H 92 IH 8 81 H233 H 8 5 IH 78 I H352 I H 69 H1359 H 783 1394 H420H 830 H 397 H 26 H 8 9 H 08 H 385 H 798 H 4 H1373H1839 H 396 H 375 H 828 H13 6 H 393 H309 H 382 H1695

H 435 H120 H 757 Dd5bH146 H 399 H 2

Dc4b H 457H 5H 89

1 48

QADRI ET AL .

FIG . 8 . Location of insertion mutations, antibody-resistant mutations, and site-specific mutations in cysteine residues of HSV-1 gB relative tothe predicted membrane orientation and secondary structure of the molecule . This figure is an amplification of the one published by Pellet et al.(1 985b) . a-Helical regions are indicated as helices, (3-sheets are indicated by zig-zags, and chain direction changes were drawn at predicted0-turns . The major helical, sheet, and turn domains were numbered from the amino terminus and prefixed with the letters H, 5, and T,respectively . Amino acids are numbered in 100s beginning at the amino acid predicted to follow the signal peptidase cleavage site . Themembrane is indicated by the stippled band . A standard one-letter amino acid code was used throughout . Numbered arrows indicate thefollowing mutants . Antibody-resistant mutants : (1) RI 392, (2) R1397, (3) R126, (4) R1375, (5) R1435, and (6) R233 (Kousoulas etal., 1988 ; Follettet al., 1985b). Point mutants in cysteine residues Cys-5 and Cys-6 . Linker-insertion mutants in the ectodomain : (7) gB-(1-475-Lk391), (8)gB-(1-475-Lk413), (9) gB-(Lk479), (10) gB-T!<600), and in the intracellular domain : (11) gB-(Lk810) .

assays showed that epitopes in neutralizing domainsD1a, D1 b, and D1c at the amino terminus, D2a, D2b,D2c, and Ma in the midregion, and Ddba proximal tothe transmembrane region of gB are exposed on thesurface of the molecule (Table 1, Fig . 7) . This surfaceconfiguration of the epitopes that bind neutralizing anti-bodies supports their functional role in infection . Our

studies on point mutants in gB indicate that cysteineresidues mapping in a functional domain of the gB mol-ecule play an important role in the folding of the regionand in maintaining its structure . Studies from anotherlaboratory recently mapped a neutralizing region be-tween residues 241 and 441 on gB and suggested thatthis region is involved in the rate of virion penetration

and spread to neighboring cells (Highlander et al.,1988) . This region overlaps the major neutralizing re-gion that we have reported, which contains the D2 andD3a domains (Fig . 7) (Kousoulas et al_ 1988 ; Pellets etal., 1 985b ; Pereira et al., 1989) . We found that the po-tent neutralizing activity displayed by antibodies thatrecognize this region precludes infectivity by wild-typevirions (Kousoulas et al., 1984, 1988 ; Pellett et al.,1985b). Experiments to determine whether these anti-bodies prevent entry or spread of virus to neighboringcells are in progress .

Mutations that affect the transport of gB

Requirements for the transport of viral glycoproteinsare varied and may be both structural and conforma-tional (see review, Rose and Doms, 1988) . One type ofstructural signal is composed of residues encodedwithin the primary amino acid sequence ; these signalsfacilitate membrane insertion and anchoring as well aslocal folding of monomers . Other structural signals in-clude the location of N-glycosylation sites (Machamerand Rose, 1988a,b) and the position of cysteine resi-duesforming disulfide bonds that promote thejuxtapo-sition of residues on the molecule (Guan et al., 1985 ;Machamer et al., 1985) . Conformational signals arediscontinuous and depend on the tertiary structure ofthe molecule . Conformation-dependent signals on gly-coproteins of influenza hemagglutinin (HA) and vesicu-lar stomatitis virus (VSV) G include the local folding ofmonomers and the overall configuration of the assem-bled oligomers (Copeland et al., 1986; Kreis and Lo-dish, 1986). These surface features depend largely onthe strategic location of glycosylation sites and the po-sition of cysteine residues that form intramolecularbonds .We have begun to understand the configuration of

the ectodomain of HSV-1 gB and the signals that pro-mote cell-surface anchoring of this glycoprotein by ana-lyzing the antigenic properties and transport of variousmutants. Two main points were demonstrated by ourstudies. First, the hydrophobic transmembrane regionis required for the stable association of gB with thecell-surface membrane . Second, perturbations of con-formational epitopes on the surface of the moleculeprofoundly affect the processing of gB through theexocytic pathway .

Deletion mutants in the carboxy terminus . Six con-structs of HSV-1 gB that lacked the transmembranehydrophobic sequences were released from cells andwere not anchored on the surface membrane . Thetruncated forms, gB-(1-393), gB-(1-475-Lk391), gB-(1-475-Lk413), and gB-(1-600), stained with an intracellu-lar pattern that suggests localization in the endoplas-

EFFECT OF MUTATIONS -N HSV- gB 149

mic reticulum . Pulse-chase experiments showed thatthe processing of these mutants differs from that ofwild-type gB and that only the more slowly migratingforms are released from cells (D . Navarro, I . Qadri, andL. Pereira, manuscript in preparation) . Longer deriva-tives, gB-(1-690) and gB-(1-690X849), which formeddimers but lacked the hydrophobic transmembranesequence, were also released from cells and were notstably associated with the surface membrane of trans-fected cells as compared with the wild-type molecule .

Insertion mutants in the ectodomain of intact gB . Inanalyzing the linker-insertion mutants in intact gB, wefound that aberrant processing and lack of cell-surfacetransport correlated with the loss of discontinuous epi-topes (Table 2) . In particular, mutant gB-(Lk479)formed dimers but lost several antigenic domains . Thismutant was retained in the endoplasmic reticulum andfailed to undergo translocation to the surface mem-brane . In contrast, gB-(Lk600), which retained the anti-genic and structural properties of the wild-type mole-cule, was transported to the cell surface . Analysis ofthe position of the linker at residue 479 (Fig . 8, No . 9)relative to the secondary structure of gB indicates thatit maps in t3-sheet 5, proximal to a-helix 11 and anN-glycosylation site at position 459 . One explanationfor the drastic effect of a mutation in this region is thatthe insertion may eliminate use of the glycosylationsite, alter the local folding of the molecule, and affectthe pairing of cysteines into disulfide bonds . Studies onthe processing of this mutant show that it remains sen-sitive to endoglycosidase H and binds the glucose-regulated proteins resident in the endoplasmic reticu-lum (D . Navarro, I . Qadri, and L . Pereira, manuscript inpreparation) . Taken together, these results indicatethat the configuration of discontinuous epitopes af-fected by mutations in the region of residue 479 is criti-cal to the transport and processing of gB .

Insertion mutants in the cytoplasmic domain . Analy-sis of mutants in the carboxy terminus of HSV-1 gBshowed that insertions of several amino acids at posi-tion 810 of the intracellular domain (Fig . 8, No. 11) didnot affect the antigenic properties of the ectodomain orthe translocation of the mutants to the cell surface . Ourresults differ from those reported in a study on linker-insertion mutants in the HSV-1 gB gene (Cat et al .,1988). That study found that mutations which impairedg8 transport were located throughout the molecule,including the intracellular carboxy terminus . These re-sults are not directly comparable with ours, since themutants in that study contained multiple insertions atdifferent sites of the carboxy terminus . Mutations in thecytoplasmic domain of VSV G glycoprotein altered itstransport from the endoplasmic reticulum, but not thefolding or trimerization of the molecule, as monitored

1 50

QADRI ET AL .

with conformation-specific antibodies (Doms et at,1988; Guan et al., 1988) . The rate of VSV glycoproteinG transport to the cell surface is, in part, determined bythe intracellular carboxy terminus (Guan et al., 1988 ;Puddington et al., 1986) . It is notable that the faster-mi-grating forms of the carboxy-terminal mutants in HSV-1gB bind the glucose-regulated proteins resident in theendoplasmic reticulum (D . Navarro, I . Qadri, and L .Pereira, manuscript in preparation) . This finding sug-gests that certain forms of these mutants may beblocked in the endoplasmic reticulum . We are in theprocess of mutagenizing other sites in the intracellulardomain of gB to understand more fully its role in trans-port of this molecule .

Location of the oligomerization signals on gB

It has long been established that viral glycoproteinsform oligomers, but relatively little is understood aboutthe signals that facilitate assembly of the ectodomaininto a quaternary structure . Our studies on deletionmutants of HSV-1 gB establish two important points :that the residues contributing to the assembly of gBdimers are located in the ectodomain between posi-tions 600 and 690, and that dimerization does not re-quire the transmembrane region or the intracellularcarboxy terminus . Studies on influenza HA showedthat secreted forms of the glycoprotein lacking thetransmembrane region and carboxy terminus can becrosslinked into trimers, indicating that the deleted re-gions are not required for oligomerization (Gething andSambrook, 1982) . Recently it was also shown that thesignals for trimer formation of VSV G map in the ecto-domain of the molecule and were independent of thehydrophobic transmembrane region and of the car-boxy terminus (Crise et al., 1989) .

In this and other studies, we analyzed the antigenicproperties of mutants in HSV-1 gB and truncated formsthat lacked portions of the carboxy terminus to corre-late their surface configuration with their ability to di-merize . We found previously that derivatives 475 resi-dues or shorter failed to form dimers (Pereira et al.,1989), a result that was confirmed in the present studywith derivative gB-(1-393) . Furthermore, we showedhere that gB-(1-600), a derivative containing 125 resi-dues more than the longest mutant analyzedpreviously, also did not form dimers, whereas gB-(1-690) and gB-(1-690A849) did . These results indicatethat the residues promoting oligomerization are con-tained in the ectodomain and overlap the discon-tinuous-epitope domains DdSa and DdSb (Fig . 7). Thefinding that 12 discontinuous epitopes are formed byresidues in this region suggests that the conformationof these epitopes is dependent on dimer assembly .

In an earlier study, we analyzed HSV-1(HFEM)tsBS, amutant in gB that forms unstable dimers at the permis-sive temperature, and found that a subset of antibodiesreactive with the parent strain H FEM did not react withthe mutant (Chapsal and Pereira, 1988) . We have sincefound that these antibodies recognize discontinuousepitopes in domains D2a, D2b, and D3b . Most notablewas the loss of five epitopes we now know are formedby residues in domains that overlap the dimer-depen-dent region, DdSa and DdSb . The finding that confor-mational changes in tsB5 dimers are coincident withthese sequences supports the involvement of this re-gion in the formation of stable oligomers . Analysis ofthe properties of mutants gB-(Lk479), gB-(Lk600), gB-Cys-5, and gB-Cys-6 indicated that these glycopro-teins form stable dimers even though several epitopesformed by residues between 600 and 690 are lost (Ta-ble 2). Furthermore, mutagenesis of the cysteine resi-due at position 603 has no effect on the dimerization ofgB (I . Qadri, D . Navarro, and L . Pereira, manuscript inpreparation). These findings suggest that the signalspromoting oligomerization of gB are encoded by a sub-set of amino acids in this region . We are in the processof mutagenizing the dimer-dependent domain to definemore precisely the signals used for assembly of gBoligomers .

ACKNOWLEDGMENTS

We thank Bin Hue and Pedro Paz for excellent technical assis-tance. These studies were supported by Public Health ServiceGrants DE-08275 from the National Institutes of Dental Researchand Al-23592 from the National Institute of Allergy and InfectiousDiseases. I .Q. was supported by a CRCC fellowship from the Aca-demic Senate of the University of California, San Francisco ; C .G . by afellowship from the Generalitat Valenciana, Conselleria de Culture,Educacion y Ciencia ; and D.N. by a fellowship from the SpanishMinistry of Education and Science .

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