Antibody-resistant mutations in cross-reactive and type-specific epitopes of herpes simplex virus 1 glycoprotein B map in separate domains

VIROLOGY 166,423-431 (1988)

Antibody-Resistant Mutations in Cross-Reactive and Type-Specific Epitopes of Herpes Simplex Virus 1 Glycoprotein B Map in Separate Domains

KONSTANTIN G. KOUSOULAS,’ BIN HUO, AND LENORE PEREIRA’

Department of Stomatology, School of Dentistry, and Department of Microbiology and Immunology, School of Medicine, University of California-San Francisco, San Francisco, California 94 143

Received February 2, 1988; accepted June 7, 1988

To characterize the domains of HSV-1 glycoprotein 6 (gB), we isolated mutants resistant to monoclonal antibodies with potent neutralizing activity. Partial nucleotide sequencing of the mutations revealed that gB contains two domains comprising discontinuous and continuous amino acids that bind cross-reactive and type-specific neutralizing antibodies. Four mutations in a discontinuous domain, R1435, R233, R1375, and Ri26, contained substitutions of Tyr278 for l-b8, HisZW for Arg,, GlnZT4 for Arg,,,, and Asn 273 for Tyr,,,, respectively. Two mutations in a continuous domain, R1392 and R1397, contained substitutions of Thr,, for Ala,, and Thr,, for AsneT, respectively, and overlapped two other type-specific epitopes. Analysis of the nucleotide sequence of strain KOS showed differences from strain F at four residues proximal to the R1392 mutation and one residue proximal to the R1397 mutation, which explains the failure of HSV-1 (F)-specific antibodies to these epitopes to react with KOS. One target site for proteolytic cleavage of gB by cellular enzymes maps at the amino terminus, partially overlapping four HSV-1 -specific epitopes. Q 1988 Academic

INTRODUCTION

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB) is incorporated into the virion envelope and is a major target for humoral and cellular immune responses (re- viewed by Norrild, 1980; Spear, 1984). gB is essential for production of infectious virus inasmuch as temperature-sensitive (ts) mutants in gB have been isolated that adsorb to cells but fail to penetrate at the nonpermis- sive temperature (Deluca et a/., 1982; Little eta/., 1981; Mansetvigi et al., 1977; Sarmiento et a/., 1979). The nucleotide sequences of gB genes from HSV-1 and -2 have been published (Bzik et a/., 1984a; Pellett et al., 1985b; Stuve et a/., 1987). Recent studies demon- strated the presence of gB homologs in the genomes of other human herpesviruses, including Epstein-Barr virus (Pellett et a/., 1985a), cytomegalovirus (Cranage et a/., 1986), and varicella-zoster virus (Davidson and Scott, 1986; Keller et a/., 1986). The conserved nature of this glycoprotein indicates that gB plays a central role in the life cycle of the herpesviruses.

Phenotypes of mutants observed to date suggest that HSV-1 gB is a multifunctional protein. Syncytial (syr-r) loci map in the domain of HSV-1 gB and other genes, suggesting that gB may interact with other proteins to mediate cell fusion (Bzik et a/., 1984b; Debroy eta/., 1985; Deluca eta/., 1982; Kousoulas eta/., 1984;

’ Present address: Department of Veterinary Microbiology, Louisi- ana State University, Baton Rouge, LA.

* To whom requests for reprints should be addressed.

Little and Schaffer, 1981; Pogue-Geile et a/., 1984; Pogue-Geile and Spear, 1987; Ruyechan et al., 1979). Native gB forms dimers and therefore contains cognitive sites for itself and other proteins (Chapsal and Per- eira, 1988; Claesson-Welsh and Spear, 1986; Haffey and Spear, 1980; Sarmiento and Spear, 1979). Temp- erature-sensitive mutations in the extracellular region of gB cause defects in processing and alter the conformation of the glycoprotein (Deluca et al., 1982; Chap- sal and Pereira, 1988; Haffey and Spear, 1980; Sar- miento and Spear, 1979). A unique class of mutants resistant to neutralizing antibodies to gB has been reported (Holland et al., 1983; Kousoulas er al., 1984; Marlin et al., 1986). Two of these mutations have been mapped to a hydrophilic region of the gB molecule (Pel- lett et a/., 198513).

In this study, we isolated mutants resistant to neutralization by antibodies to gB. Partial nucleotide sequence analysis showed that four mutations clustered in a discontinuous, type-common domain between residues 273 and 298. Two HSV-l-specific mutations mapped in a continuous domain between residues 32 and 47, a region of divergence between HSV-1 and -2 glycoproteins. A subset of epitopes at the amino terminus of gB partially overlaps the site of proteolytic cleavage by cellular enzymes.

MATERIALS AND METHODS Reagents

Restriction enzymes were obtained from Boehringer- Mannheim Biochemicals (Indianapolis, IN) and New

423 0042-6822/88 $3.00 Copyright Q 1999 by Academbc Press, Inc. All rights of reproduction in any form reserved.

424 KOUSOULAS, HUO, AND PEREIRA

England Biolabs (Beverly, MA) and were used accord- ing to the instructions of the suppliers. T4 DNA ligase, Escherichia co/i DNA polymerase I large fragment (Klenow fragment), and calf intestinal alkaline phospha- tase were from Boehringer-Mannheim Biochemicals. Sequencing primers were from New England Biolabs. Deoxy- and dideoxynucleotide triphosphates were purchased from Pharmacia P-L Biochemicals (Piscata- way, NJ). Biotin- and avidin-linked antibodies (Vectas- tain ABC Kit) were purchased from Vector Laboratories (Mountain View, CA).

Strains and vectors

Plasmid vectors pUC9 an pUC19 and their host JM83 (Vieira and Messing, 1982) were obtained from Bethesda Research Laboratories (Gaithersburg, MD).

Monoclonal antibodies

Monoclonal antibodies to HSV-1 gB were derived from hybridomas that were produced by fusion of NS- 1 myeloma cells with spleen cells of BALB/c mice im- munized with extracts of HSV-l-infected cells or with immunoaffinity-purified gB, as described previously (Eisenberg et al., 1982; Pereira et al., 1980, 1981). The properties of antibodies used in this study have been published (Chapsal and Pereira, 1988; Kousoulas et a/., 1984).

Viruses, cells, and media

Virus stocks were grown in African green monkey kidney cells (Vero) or in human epidermoid carcinoma No. 2 cells (HEp-2) which were grown in Dulbecco’s minimum essential medium supplemented with 1% fe- tal calf serum. The isolation and properties of HSV-1 (F), HSV-l(KOS), and HSV-2(G) have been published (Ejer- cite et a/., 1968; Kieff et a/., 197 1; Little and Schaffer, 1981). The properties of monoclonal-antibody-resistant mutants R233, R126, and R233Bl/R126 and the procedures for their propagation and preparation of their DNA have been reported elsewhere (Kousoulas et al,, 1984; Pellett et al,, 198513). Mutants R1392B, R1397/2, R1375, R1435/8, and R1435/1 1 were derived from HSV-1 (F) stocks propagated in the presence of 5 gg/ml of 5-bromo-2-deoxyuridine from 4 to 24 hr postinfection. The mutagenized stocks were enriched for nonreactive mutants by propagation in the presence of antibody, followed by selection of nonreactive viable plaques (white plaques) in a biotin-avidin-enhanced surface immunoassay (Kousoulas et a/., 1984).

Preparation of radiolabeled infected cell extracts

HEp-2 or Vero cells were infected with 5 PFU of virus per cell and labeled with 50 &i/ml of [35S]methionine

(sp act > 400 Ci/mmol; DuPont-New England Nuclear, Inc.) from 6 to 24 hr postinfection. For immunoprecipitation tests, infected eels were extracted with 1% NP- 40 and 1% sodium deoxycholate as described previously (Pereira et al., 1980).

lmmunoassays

Procedures used for immunofluorescence, biotin- avidin-enhanced surface immunoassays, neutralization tests, immunoprecipitation, polyacrylamide gel electrophoresis, and the transfer of electrophoretically separated proteins to nitrocellulose were described previously (Braun et al., 1983; Kousoulas et al., 1984; Pereira et al., 1980). Conditions used for native polyacrylamide gels have been described (Cohen et a/., 1986; Chapsal and Pereira, 1988).

Recombinant DNA methods

The panel of DNase I overlapping subclones of the HSV-1 (F) gB gene and conditions used for marker-rescue experiments have been reported (Kousoulas et a/., 1984). For DNA sequencing, Narl sites were used to subclone Kpnl N fragments of R1392 and R1397 into the Accl site of pUC19, and Aval sites were used to subclone R1375, R1435, and R233 Kpnl N fragments.

Marker rescue

For marker-rescue experiments, intact DNA from nonreactive mutants (white plaque phenotype) was co- transfected onto rabbit skin cells with cloned DNA fragments coding for regions of wild-type gB (Kousoulas et al., 1984). Progeny virus was collected, plated at limit- ing dilution, and scored for the appearance of reactive plaques using a biotin-avidin-enhanced surface immunoassay.

DNA sequencing

All sequencing was performed by the dideoxy tech- nique (Sanger et al., 1977) with pUCl9 as a template as described in detail previously (Pellett et a/., 198513). [a-35S]dATP (sp act 500 &i/mmol, DuPont-New En- gland Nuclear, Inc.) and gradient buffer gels were fixed and dried before autoradiography (Biggin et a/., 1983; Garoff and Ansorge, 1981).

RESULTS

Characterization of monoclonal antibodies to gB

To generate mutants resistant to neutralization, we used a panel of monoclonal antibodies to gB (Chapsal and Pereira, 1988; Kousoulas et al., 1984). Table 1 summarizes the relevant properties of the neutralizing

ANTIBODY-RESISTANT MUTATIONS IN HSV-1 gB 425

TABLE 1

IMMUNOPRECIPITATION REACTIONS OF NEUTRALIZING MONOCLONALANTIBODIESTO HSV-1 (F) gB WITH DIFFERENT CELL TYPES AND VIRUS STRAINS

Epitope$ Monoclonal antibodies

Cells infected with HSV-l(F) Viruses tested *

Denatured Native HSV-1 HSV-2

HEP Vero HEP Vero F KOS G

Discontinuous H126 H233 H1375 H1435

Continuous H1392 H1397 H1396 H1839

- - + + - - + + - - + + - - + + + - + - + - + - + - + - + - + -

+ + + + + + + + + + + + + - - + - - + - - + + -

a Epitopes on gB are defined by reactions of antibodies with sodium dodecyl sulfate-denatured gB (continuous) or native gB dimers (discontinuous) (Chapsal and Pereira, in press).

*Viruses propagated in HEp-2 cells.

antibodies used in this study. lmmunoprecipitation tests were done with HEp-2 and Vero cells infected with different virus strains. Immune reactions were also performed with electrophoretically separated polypeptides transferred to nitrocellulose under native or denaturing conditions (see Materials and Methods). Epi- topes of H126, H233, H1375, and H1435 antibodies were designated as discontinuous because they failed to detect gB denatured with sodium dodecyl sulfate, reacting only with gB dimers electrophoresed in polyacrylamide gels and transferred to nitrocellulose under native conditions (Chapsal and Pereira, 1988). In con-

trast, H1392, H1397, H1396, and H1839 antibodies recognized continuous epitopes on gB dimers and on native or denatured monomers. H 1397 and H 1396 antibodies were specific for HSV-l(F) whereas H1392 also reacted weakly and H1839 antibody reacted stronglywith strain KOS. All antibodies had neutralizing activity and reacted strongly in the enhanced surface immunoassay used to select nonreactive mutants.

Characterization of antibody-resistant mutants in neutralization tests

To identify the amino acids that participate in neutralizing epitopes, we first isolated mutants resistant to

TABLE 2

PERCENTAGE NEUTRALIZATION OF MONOCLONAL ANTIBODIES REACTED WITH HSV STRAINS AND ANTIBODY-RESISTANT MUTANTS IN gB”

Monoclonal antibodies

Viruses H126

Type-common

H1375 H1435 H233 H1392

Type-specific

H1396 H1397

HSV-1 (F) 100 100 100 100 53 50 98 HSV-2 (G) 100 100 100 100 0 0 0 HSV-1 (KOS) 100 98 100 100 0 0 0 R126 0 0 100 100 100 100 100 R1375 0 0 100 100 100 100 100 R 143518 37 0 0 100 100 100 100 R1435/1 1 29 0 0 100 100 100 100 R233 100 100 100 0 100 100 100 R126/233 0 0 100 0 100 100 100 R1392 100 100 100 100 0 0 86 R1397 100 100 100 100 0 0 0

a For neutralization tests, 200-300 PFU of virus was reacted with 10 ~1 of mouse ascites for 2 hr at room temperature and then plated on Vero cells. Data are means of three separate tests.


126 233

FIG. 1. Autoradiograms of electrophoretically separated polypeptides in immune precipitates, obtained by reacting homologous and heterologous monoclonal antibodies with infected HEp-2 cells labeled with [%]methionine. Strains HSV-l(F), HSV2(G), and HSV-l(KOS) and mutants R1435/8, R1435/11, R126, R233, R126/233, and R1375, resistant to cross-neutralizing antibodies, are shown in top and middle panels. Mutants R13928, R1397, R1392, and R1397/2, resistant to HSV-1 -specific antibodies, are shown in bottom panel. Viruses are listed above each lane and antibodies are indicated above each section. gB + shows the precipitated glycoprotein.

cross-reactive antibodies H233, H1375, and H1435 and HSV-l-specific antibodies H1392 and H1397, as described under Materials and Methods. A mutant resistant to H 126 was isolated previously (Kousoulas et a/., 1984). To detect overlapping epitopes, the mutants were also reacted with heterologous antibodies (Table 2). Antibodies to discontinuous, type-common epitopes neutralized strains HSV-1 (F), HSV-1 (KOS), and HSV-2(G). Cross-reactivity tests showed that mutant R126 was resistant to H1375 antibody and mutant R1375 to H126 antibody, indicating that the epitopes were affected by either mutation. H 1435 antibody neutralized R126 and R1375 mutants, but R1435/8 and R1435/1 1 were neutralized only partially by H 126 and were not neutralized by H 1375. These results indicate that the H 126 and H1375 epitopes were affected by the R1435 mutation, but that the H 1435 epitope was not altered by the R126 and R1375 mutations. R233 was neutralized by H126, H1375, and H1435 antibodies, and H233 antibody neutralized heterologous mutants R126, R1375, and R1435.

Antibodies to HSV-1 specific epitopes also showed complex neutralization patterns. H 1392 and H 1397 antibodies neutralized strain F but not KOS, G, or mutants R1392 and R1397 selected with the homologous antibody. Cross-reactivity tests showed that antibody H1397 almost completely neutralized R1392 but that H1392 antibody failed to neutralize R1397. Although H 1396 did not select resistant mutants, the antibody failed to neutralize either R1392 or R1397. Insofar as the H 1396 epitope was altered by mutations in R1392 and R1397, the epitopes are partially overlapping.

Properties of resistant mutants in immunoprecipitation tests

The next series of experiments was done to determine whether the selected mutants in gB would bind antibodies in immunoprecipitation tests. Antibodies to discontinuous epitopes reacted with HSV-1 (F), HSV- 1 (KOS), and HSV-2(G) but generally failed to react with gB specified by the selected mutant (Fig. 1). Whereas


mutants R126, R1375, R1435, and recombinant R126/ 233 failed to react with the homologous antibodies, neutralization-resistant mutant R233 was precipitated by H233 antibody. It was of interest that H 1375 antibody reacted with mutant Rl435/8 but failed to recognize R1435/1 1, suggesting that mutations in the inde- pendent plaque isolates differed. Reactions with heterologous antibodies paralleled results of neutralization tests suggesting that the epitopes of H126 and H 1375 overlap and that the H233 epitope is separate from those of H126, H 1375, and H1435.

Antibodies to continuous epitopes also generally failed to react with gB specified by the selected mutant; the exception, R1392B, a spontaneous mutant, was precipitated by H 1392 antibody. Although repeated selection with antibodies H1396 and H 1839 failed to yield nonreactive mutants, H1839 reacted weakly and H1396 did not react with the heterologous R1392 mutant. Inasmuch as H 1839 precipitated strain KOS gB and H 1392 reacted weakly with KOS (data not shown), these epitopes may partially overlap. These results are supported by the finding that H 1839 antibody reacts weakly with R1392 and strongly with other mutants (Fig. 1).

Mapping and nucleotide sequence analyses of mutations that result in neutralization resistance

To map the approximate location of antibody-resistant mutations in gB, we performed marker rescue experiments by cotransfecting intact mutant DNA and wild-type DNA fragments with staggered deletions in gB, as described previously (Kousoulas et al., 1984). Mutations R233, R1375, and R1435/8 were rescued by plasmids pRB2021 and pRB2094, which overlap by 377 bp. DNA fragments encoding the portion of the gB sequence from amino acids 147 to 454 were cloned and sequenced. The mutation in R1375 was a G to A base change resulting in the substitution of Gln for Arg at residue 274, and the mutation in R1435 was a C to T base change resulting in the substitution of Tyr for His at residue 278 (data not shown). The mutation in R233, a G to A base change, resulted in the substitution of His for Arg at residue 298.

The R1392 and R1397 mutations were rescued with plasmids pBR2085 and pBR2069, which contain DNA fragments overlapping by 180 bp, encoding residues -4 to +57. DNA fragments that encode gB residues -26 to +91 in the amino terminus were cloned and sequenced. The mutation in R1392 was a G to A base change resulting in a substitution of Thr for Ala at residue 32, and the R1397 mutation was an A to C base change resulting in the substitution of Thr for Asn at residue 47. The sequencing results are summarized in Table 3.

TABLE 3

NUCLEOTIDE SEQUENCE AND PREDICTED AMINO ACID CHANGES IN CON- TINUOUS AND DISCONTINUOUS EPITOPES OF ANTIBODY-RESISTANT Mu- TANTS IN gB

Epitopes’

Antibody- resistant mutants

Changes in nucleotide sequence

Predicted amino acid

substitutions

Discontinuous

Continuous

R233 CGC + CAC Amg8 -+ Hi% R1375 CGG + CAG Am4 + Glh R1435 CAC + TAC Hb8 + Tyr278 R126/Blb TAC + AX TyrzT3 - Ash

AGC -, ACC SerzB3 + Thrza3 R1392 GCC + ACC Ah + Thrs2 R1397 AAC -P ACC AS%, + Thr4,

a Epitopes on gB are defined by reactions of antibodies with sodium dodecyl sulfate-denatured gB (continuous) or native gB dimers (discontinuous) (Chapsal and Pereira, in press).

* Mutation previously mapped (Pellet et al., 1985b).

Comparison of the amino acid sequence of gB encoded by strains F, KOS, and G

The observation that H 1392, H 1396, and H 1397 antibodies to continuous epitopes reacted with strain F but not KOS or HSV-2(G) led us to compare the predicted sequence of the amino terminus of gB specified by these strains and by mutants R1392 and R1397. The nucleotide sequence and predicted amino acid sequence of gB specified by strains F and KOS have been published (Bzik et al., 1984a; Pellett et a/., 1985b). Comparison of the amino acid sequence of HSV-2(G) (K. Kousoulas, M. Arsenakis, and L. Pereira, manuscript submitted for publication) with the published sequence of strains HG52 and 333 (Bzik et a/., 1986; Stuve et a/., 1987) showed no differences in this region of the molecule. The aligned HSV-2(G) sequence car- ried a deletion within the signal sequence from amino acids -27 to -21. Analysis of the gB sequence of strain KOS showed differences from strain G in three residues and from strain F in five residues, all proximal to the substitutions found in the R1392 and R1397 mutants (Fig. 2). The mutation in R1392 resulted in an Alas2 to Thr,, substitution, whereas KOS exhibited changes from F in residues Alap to ProZ8, ProZ9 to Leu,, , Pros, to Ala,, , and Thr,, to Pros,. The R1397 mutation, an Asn4, to Thr,, substitution, was proximal to a residue difference in KOS at Prod9 to Thr,,. These differences appear to overlap the H 1392, H 1396, and H 1397 epitopes and may be responsible for the failure of these antibodies to react with KOS. The finding that H1839 reacted with KOS and weakly with mutant R1392 (Fig. 1) suggests that this antibody recognizes residues common to strains F and KOS that are proximal to the H 1392 epitope.

428 KOUSOULAS, HUO. AND PEREIRA

n*“-ll108)

HSV- 1 (F)

IiSV-Z(G)

MS”-1 IXOSI

HSV-l(F)

HSV-2(G)

em-1 (XCS)

HSV-l(F)

HSV.2(0)

SSV-1 ,IOSl

HSV-1 (F)

HSV.2(0)

ss”-l(Kos~

HSV-1 (F)

WV.?(G)

-29 ‘1’ ‘f” .;=ty yP

Met-arggln-gly-ala-ala- .._.. - arg-gly-cys-arg-trp-phe-r;al-;al-~~ala-leu-leu~~-~u-thr-leu~gly I I I IIll I

-1 l l

val-leu-val-ala-ser-a~-ala-pro-s~r~s~r-pro-gly-lhr~pr~gl~val-ala~ala~ala~lhr+ln-ala-ala-asn-gly- I I I I

9/y A I

Ita aa Ill aa 111 IIs VII

pro l=” “I El Pro

I I gly-pro-ala-f~pr~-a~-pro-pro~a~p~~g~y-pro-ala~p~-lhr~g~~~pl~~s-~~~s-~~~lys-

SW er9 pm WI SW lhr Irr a w l r9 Ihr lye

lys-pro-lys-asn-pro-pro-pro-pro-a~g-pro-~la-~l~a~p-asn-ala-thr-val-ala-a~~~-his-ala-~hr-leu- I I II I I

w ghl a* Ihr Pro pro .sp sls Vel

47

arg-glu-his-leu-arg-asp-ile-lys-a~glu-asn-t~r-as~ala-a~phe-tyr-val~s-pro- I I

II@ !JlU VII sls 9ln

FIG. 2. Comparison of the predicted amino acid sequence of the region coding for -29 to +87 residues of gB specified by HSV-l(F) (Pellett er a/., 1985b), HSV-1 (KOS) (Bzik et al., 1984) and HSV-2(G) (Kousoulas et al., manuscript in preparation). Amino acids from -29 to -1 contain the signal sequence. Residues in the KOS sequence (top line) and the G sequence (bottom line) are compared with those of strain F (middle line). Residues that differ are indicated and missing residues are represented by (. . .). Mutations in R1392 and R1397 are also indicated (boxes). N- glycosylation site, shaded box.

Comparison of the amino-terminal sequence of gB from strain G with that of strain F showed differences in 37 residues, rendering the amino terminus of the molecule highly type-specific. Results of this study showing that continuous epitopes of four type-specific antibodies mapped within the amino terminus of gB were supported by transient expression of truncated derivatives (L. Pereira, K. Kousoulas, M. Ali, B. Huo and T. Banks, manuscript in preparation) and by analysis of the immunologic properties of gB made by a HSV-1 x HSV-2 inter-typic recombinant in this gene (Kousou- las et al., manuscript submitted for publication).

Reactivity of antibodies with HSV-1 gB made in Vero cells and HEp-2 cells

We previously reported that HSV-2 gB undergoes cleavage in Vero cells into fragments with greater mo- bilities, and that a HSV-2-specific antibody failed to react with cleavage products made in Vero cells (Pereira et a/., 1981, 1982). To determine whether HSV-1 -specific antibodies react similarly, we infected Vero and HEp-2 cells with strain F or G, precipitated extracts with antibodies, and subjected the precipitates to electrophoresis in denaturing polyacrylamide gels. Results of these experiments are summarized in Table 1. Type-

specific H 1392, H1396, H 1397, and H 1839 antibodies reacted with HEp-2 cells infected with strain F but not G, and failed to react with extracts of infected Vero cells. The finding that cleavage disrupts the reactivity of four antibodies, and that the epitopes of these antibodies are type-specifc and map in the amino terminus of the molecule, indicates that one cleavage site is within the first 50 amino acids of the amino terminus and is partially overlapped by these epitopes.

DISCUSSION

In this study, we report the mapping of two domains within HSV-1 (F) gB that contain epitopes of potent neutralizing monoclonal antibodies. On the basis of these results, we have constructed a topographical model of the domains of gB (Fig. 3). The model maps epitopes of four antibodies to a type-specific domain of continuous amino acids 32 to 47 and epitopes of four antibodies to a type-common, discontinuous domain between amino acids 273 and 298. Epitopes of eight indepen- dently derived monoclonal antibodies clustered in the hydrophilic region of gB, indicating that these domains are highly antigenic and accessible on the surface of gB oliogomers. It remains to be determined whether


TYPO Sp*clflc Typa Common Contlnuour Dlscontlnuour

Neut Neut Rate of

1 2 Entry Syn -

coon 552 6 5 7 amino acids

-*.. -. *. --.*

--. -+. Epltopes Epltopes Domain Domain 1

1II

H1392 H233 Ii1397 H126 H1396 H1375 H1839 H1435

FIG. 3. Schematic representation of a topographical map of HSV-1 gB. Neutralizing domains, continuous type-specific (1) and discontinuous type-common (2). are labeled by ellipses. Amino acids are indicated by numbers. Map locations for rate of entry and syncytia (syn) formation on strain KOS are marked (vertical lines) (Bzik era/., 1984b). Transmembrane anchor domain (box) and amino (NH,) and carboxy (COOH) termini are shown (Bzik et al., 1984a; Pellet et a/., 1985b). Epitopes of antibodies mapping in neutralizing domains are listed.

additional epitopes map in these regions orwhether gB specifies other neutralizing domains.

Discontinuous cross-neutralizing epitopes

We found that single amino acid changes in discontinuous epitopes of three cross-neutralizing antibodies, H1375, H1435, and H233, map proximal to the H 126 epitope (Pellett et al., 1985b). Cross-reactivity profiles indicated that H 126, H 1375, and H 1435 epitopes partially overlap each other but that H233 is separate. We previously reported that the R126 mutant contained two altered residues at 273 and 283 (Table 3) (Pellett et al., 1985b). Repeated analyses of the nucleotide sequence in this region showed that residue 283 is the same as in the parental strain and can be excluded from contributing to the loss of H126 reactivity (K. Kousoulas and L. Pereira, unpublished results). The remaining R126 mutation at residue 273 is proximal to the R1375 mutation at residue 274, consistent with the evidence that these epitopes overlap. Since altered residues causing loss of reactivity to antibodies H 126, H 1375, H 1435, and H233 are part of assembled epitopes, it is possible that residues distal to the amino acid substitutions mapped may contribute to the nonreactive phenotype. Using transient expression of truncated forms of gB, we found that amino acids required for proper folding of the discontinuous domain are distal from the epitopes mapped (L. Pereira et a/., manuscript in preparation). Nevertheless, the present results and other studies indicate that amino acid changes abolishing antibody binding are crucial to the cognitive site (Wiley and Skehel, 1987).

Continuous HSV-1 -specific epitopes

In this study, we isolated mutants nonreactive with two antibodies, H 1392 and H 1397, that recognize continuous epitopes on HSV-1 gB. Other amino acids comprising these epitopes were expected to be proximal to the mutated amino acid in the primary sequence. Continuous epitopes are most likely to contain highly hydrophilic residues in P-turn structures (Rose, 1978; Eisenberg eta/., 1985). This prediction was used to locate a continuous neutralizing epitope that mapped at the amino terminus of HSV-1 gD (Cohen et a/., 1984). DNA sequence analysis of resistant mutants R1392 and R1397 showed that these mutations were located within a hydrophilic region intersecting a P-turn structure extending from residues 16 to 58 (Pellett et a/., 198513). Further evidence that this region of gB contains continuous epitopes comes from the finding that H1396 and H 1397 antibodies react with a synthetic peptide of HSV-l(F) gB containing residues 31 to 53 (C. Christian and L. Pereira, manuscript in preparation). Taken together with the current finding that the R1392 mutation is at residue 32, it appears that most of the contiguous residues comprising epitope H1392 lie to the left of the H 1396 and H 1397 epitopes. The extent of overlap within the continuous domain of gB remains to be determined. To resolve this problem, we are using a set of overlapping synthetic peptides of gB to further define the boundaries of these epitopes.

Divergence of the gB nucleotide sequence and antigenic specificity

Recent reports on the nucleotide sequences of HSV- 1 and HSV-2 gB genes showed that the amino end of the molecule exhibits a high degree of divergence between these types (Bzik et al., 1984a, 1986; Pellett et al., 198513; Stuve et al., 1987). By partial nucleotide sequence analysis of the gB gene, we found that 37 residues differed in the amino terminus of strains F and G (Kousoulas et al., manuscript in preparation). Intertypi- callydivergent domains that contained hydrophilic residues in a P-turn structure would be likely cognitive sites for type-specific antibodies. Comparison of the nucleotide sequence of HSV-1 strains F and KOS (Fig. 3) re- veals that all but four residue differences are concen- trated in the first 76 amino acids. Results of the present study showed that epitopes of four type-specific antibodies map in this domain of HSV-1 gB, indicating that the amino terminus contains residues responsible for type-specific and strain-specific antigenicity. Analysis of R1392 showed a substitution at residue 32 mapping proximal to the residues (28, 29, 31, and 37) in strain KOS that differ from strain F. These differences may account for loss of H 1392 reactivity with KOS. Like-


wise, strain KOS exhibits a change at residue 49, proximal to the R1397 mutation at position 37. The failure of H1397 to react with strain KOS indicated that ProJ9 is a participating residue in the H1397 epitope. The observation that H 1392 antibody failed to neutralize the R1397 mutant suggests that this mutation affects the partially overlapping H 1392 epitope. If this is true, then one or more of the amino acids at positions that differ between strains F and KOS may participate in the H 1392, H 1396, and H1397 epitopes. Analyses of additional mutants selected with these antibodies should further define the residues that comprise each epitope.

Proteolytic cleavage of gB in Vero cells

We reported that HSV-2 gB made in Vero cells specified different immunological and electrophoretic properties from gB made in HEp-2 cells, as a result of cleavage by cellular enzymes (Pereira et a/., 1981, 1982). The observations that cleavage precluded the reactivity of a subset of neutralizing antibodies and that the cleavage fragments were glycosylated suggested that they were derived from the extracellular region of the molecule. In the present study, we found that H1392, H1396, H1397, and H1839 antibodies, whose epitopes map at the amino terminus of gB, failed to precip- itate the Vero cell products. This result is consistent with the notion that the cleavage sites are located at the amino terminus of the molecule and partially overlap these epitopes.

ACKNOWLEDGMENTS

These studies were aided in part by Public Health Service Grants DE-08275 from the National Institute of Dental Research and Al- 29592 from the National Institutes of Allergy and Infectious Diseases and by a grant from the Academic Senate Committee on Research Support of the University of California San Francisco. K.G.K. was a postdoctoral trainee in the Public Health Service Training Program in Sexually Transmitted Diseases (P32-AC07234).

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Antibody-resistant mutations in cross-reactive and type-specific epitopes of herpes simplex virus 1 glycoprotein B map in separate domains

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