Antigenic variation (mar mutations) in herpes simplex virus glycoprotein B can induce temperature-dependent alterations in gB processing and virus production.

0022-538X/86/070142-12$02.00/0

Copyright C) 1986, American Society for Microbiology

Antigenic

Variation (mar Mutations) in Herpes Simplex Virus

Glycoprotein B Can Induce

Temperature-Dependent

Alterations in

gB Processing and Virus

Production

S. D. MARLIN,'t S. L. HIGHLANDER,' T. C. HOLLAND,2t M. LEVINE,3 ANDJ. C.

GLORIOSO2*

The GraduateProgram in Cellular and Molecular Biology,' Department of Microbiology and Immunology and Unitfor LaboratoryAnimal Medicine,2 andDepartmentofHuman Genetics,3 University ofMichigan Medical School,Ann Arbor,

Michigan48109-0010

Received 27 December1985/Accepted 25 March 1986

Monoclonal antibody-resistant (mar) mutants altered in the antigenicstructure of glycoproteinB (gB) of herpes simplex virustype 1, strain KOS-321,were selected byneutralization witheachof six independently

derived gB-specific monoclonal antibodies. Analysis ofthereactivitypatternsofthesemarmutantswithapanel

of 16virus-neutralizingmonoclonal antibodies identifiedatleastfivenonoverlapping epitopesonthisantigen,

designatedgroupsIthrough V. Multiplemarmutationswerealso introducedintothe gBstructural geneby

recombination and sequential antibody selection to produce a set ofmarmutants with double, triple, and quadruple epitope alterations. Group II (B2) and group III (B4) antibodies were used to select the corresponding mutants, mar B2.1 and mar B4.1, which in addition to carrying the mar phenotype were

temperature sensitive (ts) for processing of the major partially glycosylated precursor of gB, pgB (Mr =

107,000),tomaturegB (Mr = 126,000)andshowed reducedlevelsofgBonthe cellsurfaceathightemperature (39°C). These mutantswerenot, however,tsforproductionofinfectious progeny. Arecombinantvirus,mar

B2/4.1, carryingboth of thesealterations wastsfor virusproductionand failed toproduceand transportany detectablematuregBtothecell surfaceat39°C. Rather, pgB accumulatedintheinfected cell. Revertants of the ts phenotype, isolated from virus plaques at 39°C, regained the B2 but not the B4 epitope and were

phenotypically indistinguishablefrom themarB4.1 parent. Finally,itwasshownthatgroupII(B5)andgroup

III(B4) antibodiesfailedtoimmunoprecipitate pgB (39°C)produced bytsgBmutantsofherpes simplex virus type 1whichwerenotselected withmonoclonal antibodies.Takentogether, ourfindingsindicate that(i)mar

mutations can alter antigenicas well as otherfunctional domains ofgB, namely, the domain(s) involved in processingandinfectivity,and(ii) groupIIandgroupIIIepitopeslie withinanessential functional domain of

gB which isa targetfortsgBmutations.

Herpes simplex virus (HSV) encodes at least six

glyco-proteins designatedgB,gC, gD, gE, gH, andgG (3, 34, 36).

Although many of the HSV glycoproteins were first

de-scribedover 10 years ago(34), littleinformation is available concerning their functionin virusreplicationandinfectivity.

It is clear that they are incorporated into virion envelopes and are transported to the cell surface membrane during infection (36). Because of their exposed position on these membranes they play a crucial role in virus infectivity, pathogenesis, and the immunobiology ofinfection (12, 21,

24, 25, 35).

Thusfar, gB is the only glycoproteinfor which tempera-ture-sensitive(ts)mutantshavebeendescribed (7,13, 22,30) identifying gBas anessentialvirus component. Although ts

gB mutants grow normally and produce phenotypically

normal gB at permissive temperature (34°C), they produce very little fully glycosylated gB atthe nonpermissive tem-perature(39°C). Rather,alower-molecular-weight form(Mr

= 107,000 [107K])accumulates in thecytoplasm, aproduct which is not exposed on the cell surface as is the mature

species(Mr = 126,000) (30). In contrast tothematureform,

the 107K species is endoglycosidase H sensitive and is

* Correspondingauthor.

t Presentaddress: Dana Farber CancerInstitute, Harvard Uni-versity Medical School, Boston, MA 02115.

t Present address: Department ofMicrobiology and Immunol-ogy, Wayne State MedicalSchool, Detroit, MI 48201.

similar in sizeto themostabundantgB precursor, pgB(18,

37). It appears that ts gB mutant virus produced at39°C is

enveloped and will attach to host cells (22, 30). However,

these mutantsfailtopenetrate host cell surface membranes

unless the virion envelopes are artificially fused to the cellular plasma membranes with polyethylene glycol (22, 30). These

findings

were

interpreted

to mean that gB is essentialtothe processof viruspenetration (22, 30). Related

toitsfunctioninviruspenetration, gBhas been showntobe involved infusingcellmembranes, therebyformingsyncytia

(7, 20). One of thefoursynlociin HSVtype1 (HSV-1)has been physically mapped to the coding sequence for the

cytoplasmic domain of gB (7, 20).

The HSV glycoproteins also have been shown to be the

principal antigensresponsiblefor the induction ofimmunity

to HSV infection (6, 35). They induce both humoral and cell-mediated protective immune responses and

provide

targetantigens for nonimmune defense mechanisms suchas natural killer cells (1, 2, 4, 5, 9, 13, 24-27, 32, 33, 39). The

characterization of the antigenic structure of the HSV

gly-coproteinsis essentialto ourunderstandingof the

immuno-biology of HSV infection. Studies along these lines have beenaidedbyusingHSVglycoprotein-directed monoclonal

antibodies(8, 15, 16,20, 23,28).Thesehighlyspecific probes

have been used to catalog distinct antibody-binding sites

(epitopes) andorganizethem into antigenic sites composed

ofoverlapping epitopes (10, 23).

142

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Our laboratory has used virus-neutralizing monoclonal

antibodiesto select aseries of antigenic variants in each of

the HSV-1 glycoproteins gC, gB, and gD; the resulting

strains are referred to as monoclonal antibody-resistant

(mar) mutants (15). The identification of unique reactivity patternsofmar mutantswithpanels ofmonoclonal antibod-ies operationally defines distinct epitopes (23). mar

muta-tions alterthe sequence orconfiguration of aminoacids with which an antibody reacts. Thus far, all mar mutations studied have been shown by marker rescue to map physi-cally to their respective glycoprotein structural genes (16,

28). The identification ofthesemutationsby DNA

sequenc-ing indicatesthey resultfrom point mutations (28;B. Wu, F. Homa, L. Holland, D. J. Dorney, J. C. Glorioso, and M.

Levine, manuscript in preparation). In this report, we

de-scribe the useof monoclonal antibodiesand mar mutants to extend our understanding of the antigenic structure of

HSV-1 gB. In addition, we provide evidence that mar

mutations in gB appear toalter this antigen athigh temper-ature,which results in defectsingBprocessingandfunction

in virus replication.

MATERIALS ANDMETHODS

Virus strains. Wild-type HSV andantigenic variantswere grown andtiterswere determinedon Africangreen monkey kidney (Vero) cells at 37°Cby infection at low

multiplicity

(15). Plaque-purified isolates of wild-type HSV-1

(strain

KOS) and HSV-2 (strain 186), designated KOS-321 and

186.111, respectively, were used in all

experiments.

The KOS mutant, tsF13, wasisolatedin M. Levine's

laboratory

by in vitro mutagenesis ofthe EcoRI F

fragment

and has

been described previously (13). Also

employed

were ts B5

(HSV-1, strain HFEM) and ts J12

(HSV-1,

strain

KOS),

kindly provided by Patricia Spear

(University

of

Chicago)

andPriscilla Schaffer(Harvard

University), respectively.

All three tsgB mutants were tsforvirusgrowth and

processing

of pgB to gB (13, 22, 30). The ts mutants were grown and

titerswere determinedat 34°C (13).

Cell cultures and media. Human

embryonic lung (HEL)

and Vero cells were grown and maintained in

Eagle

mini-mumessential medium(GIBCO

Laboratories,

Grand

Island,

N.Y.) supplemented with nonessential amino

acids,

10 mM

HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid), and 10% fetal calfserum

(GIBCO Laboratories).

The

BALB/c myeloma cell line, P3-X63-Ag8.653, and the

my-eloma-spleen cell hybridomas were grown in

supplemented

Dulbecco modified Eagle medium (GIBCO

Laboratories)

containing 20% fetal calf serum

(Sterile

Systems, Inc.,

Logan, Utah),asdescribed previously (15).

Production of hybridomas and polyclonal antisera. The

procedures used fortheproduction of hybridomas

secreting

neutralizingmonoclonal antibodies specificfor HSV-1 have been described in detail elsewhere (15).

Briefly,

BALB/c

micewereinfected

intraperitoneally

with 2 x 107PFUoflive

KOS-321 virusonday1and boostedon

day

13with

108

PFU

of UV-inactivated virus. On

day 16,

immune

spleen

cells and

P3-X63-Ag8.653 myeloma cells were fused using

polyethyl-eneglycol.

High-titer ascites fluid

preparations containing

monoclo-nal antibodies were produced as

previously

described(23).

Antibodies fromclarified ascites fluid were concentratedby

ammonium sulfate precipitation and characterized for

monoclonality byisoelectricfocusingasdetailed earlier(23).

Monoclonalantibody isotypes were determined

by

enzyme

immunoassay with

subclass-specific

antisera

using

a

MonoAb-ID EIA Kit

(Zymed Laboratories,

San

Francisco,

Calif.).

In some

experiments,

a

pool

of

equal

volumes of

antibodies

B2, B3,

B4,

and B6 was used. The monoclonal antibodies3S,

24S, 25S, 30S, 33S, 40S,

and 61Swere

kindly

provided

by Martin

Zweig (National

Cancer

Institute,

Frederick, Md.).Theseantibodieswere

produced

from mice

immunized with HSV-1

(strain 14012)

and have been

char-acterized

by

Showalteret al.

(33).

To produce

HSV-1-specific antisera,

groups of three

BALB/c mice were immunized

by

intraperitoneal

infection with various dosesofvirus

ranging

from

102

to

106

PFU per mouse.Themicewerebled fromtheretroorbital

cavity

at3, 5, 7,and 10days after

immunization,

andserafromeach

day

were pooled.

Virusneutralization assays.

Wild-type

virus and

antigenic

variantsweretestedfor

reactivity

withmonoclonal antibod-ies in 50%

plaque

reduction microneutralization assays as

reported

previously

(23). Neutralization titers of

concen-trated ascites fluid stockswere

expressed

asthe

reciprocal

of

the

highest

dilution which reducedastandardized virus

input

(20 to 30 PFU per

well) by

at least 50% relative to

non-neutralizedcontrols.

Mouse antiserawere

assayed

for

virus-neutralizing

activ-ity

in

large

scale assays.

Equal

volumes

(0.5

ml)

of

serially

diluted

antibody

and virus stock

(2,000

PFU/ml)

weremixed andincubated asdescribed above. Titers of

surviving

virus were then determined on Vero cell

monolayers

in 60-mm

dishes,

in which

approximately

200

plaques

could be

counted in the unneutralized controls. Results were then

expressed

as percent neutralization at each

antibody

dilu-tion.

Enzyme immunoassays. The

binding

of monoclonal anti-bodiesto

glutaraldehyde-fixed,

virus-infectedHELcellswas

detected

using

horseradish

peroxidase-conjugated

goat

anti-mouse

immunoglobulin

G

(IgG)

inanenzyme

immunoassay.

This assay has beendescribedindetail

by

Marlinetal.

(23).

A405valuesweredetermined withaTitertekMultiscan

(Flow

Laboratories,

Rockville, Md.).

The

immunoperoxidase

"black

plaque"

assay was

performed

as

previously

de-scribed

(16).

Briefly,

Vero cell

monolayers

in 60-mmdishes were infected with 20 to 200 PFU of

virus,

glutaraldehyde

fixed after

plaque

formation,

and incubatedwith

gB-specific

monoclonal antibodies.

Next,

monolayers

were incubated

with horseradish

peroxidase-conjugated

goat anti-mouse

IgG.

Subsequent

treatment with

4-chloro-1-napthol

pro-ducedablack

precipitate

on

plaques

towhich

gB

antibodies

werebound.

Radiolabeling,

immunoprecipitation,

and

electrophoresis.

HEL cells were infected with

wild-type

KOS-321 virus or

antigenic

variants at a

multiplicity

of 10 and radiolabeled

with 40

,Ci

of

[35S]methionine

per ml from 4 to 18 h

postinfection

at either 34 or

39°C.

Nonidet P-40extracts of

radiolabeled infected cells were

immunoprecipitated

with a

pool

of

gB-specific

monoclonal

antibodies, electrophoresed

on 7%

polyacrylamide

slab

gels

cross-linked with

diallyl-tartardiamide,

and

fluorographed

asdetailed

previously (16).

Isolation of

single

and

multiple antigenic

variants.

Anti-genic

variants of

wild-type

HSV-1

(KOS-321)

resistant to neutralization

by single

gB-specific

monoclonal antibodies in the presenceof rabbit

complement

were isolated

essentially

as described earlier

(15).

Isolates of the virus

surviving

neutralizationwere

plaque

purified

threetimes and tested for neutralization in the 50%

plaque

reduction assay described above. Those

antigenic

variants which

qualified

were

desig-natedmonoclonal

antibody-resistant (mar)

mutants

accord-ing

to

previously

definedcriteria

(15).

Multiple

marmutants

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wereselected sequentially byusingtwoormoremonoclonal

antibodies from virus produced during mixed infections with singlemar mutants(see Results).

Some antigenic variants were isolated from stocks of

KOS-321 mutagenized with 5-bromo-2-deoxyuridine as

re-ported earlier (15). Briefly, viruswas grownin Vero cellsat

amultiplicity of infection (MOI) of 5 for 24 h in thepresence

of2.5 ,ug of5-bromo-2-deoxyuridine perml. Before

mono-clonal antibody selection, the mutagenized virus was

pas-sagedonVerocells atan MOI of 0.01 for 24 h toeliminate phenotype mixing ofmutant and wild-typeglycoproteins in thesame virion envelope.

Isolation of ts+ revertants from ts double mar mutants. Revertantvirusesnolongertsforplatingwereisolated from

thedoublemutant marB2/4.1 by passageatthe nonpermis-sivetemperature. Vero cell monolayerswere infectedatan

MOI of 0.01 and incubated at 39°C until cytopathic effect

wasgeneralized. For isolation of therevertant,R3marB4.1, two rounds of low-MOI passage at 39°C were done. The appearance of putative revertants was identified by

deter-mining titers of the resulting virusat34 and39°C. Individual plaqueswere picked from the plates incubatedat 39°C, and small stocks were grown in Vero cells at 34°C. The virus isolateswerethenplaquepurified three timesandtested for

plating efficiency and virus yield at permissive and nonpermissive temperatures.

RESULTS

Characteristics of monoclonal antibodies. Apanel of mono-clonal antibodies was produced for use as probes of the

antigenic structureofgB. Nine hybridomas secreting virus-neutralizing monoclonal antibodies were derived from six

independent fusions using spleen cells from BALB/c mice immunized by infection with wild-type HSV-1 (KOS-321)as

describedpreviously (23). As demonstrated earlier (15),the antibodies were determined tobe specific forgB by

immu-noprecipitationof[35S]methionine-labeled gB from Nonidet P-40lysates of HSV-1-infected cells (datanot shown). The derivation andcharacteristics of the monoclonal antibodies arelisted inTable 1. All oftheantibodies neutralized HSV-1 tohigh titers in thepresenceof rabbitcomplement,butonly antibody B4 produced significant neutralization in the ab-senceofcomplement. Six oftheantibodies(B2, B4, B5, B7, B8, and B9) were cross-reactive with HSV-2 and thus

[image:3.612.320.560.86.187.2]

recognized epitopes shared by thetwo strains tested. TABLE 1. Characteristics ofgB-specificmonoclonal antibodies

Neutralization titer Monoclonal Fusion Isotype HSV-1(KOS-321) HSV-2

antibody no. HSV_____2_

+a _b (186.111)c

B1 1 IgG3 2,560 <10 <10

B2 3 IgG2b 163,840 <40 40,960

B3 4 IgG3 5,120 <40 <40

B4 4 IgG3 2,560 1,280 640

B5 5 IgG3 81,920 <160 5,120

B6 7 IgG2b 81,920 <160 <160

B7 6 IgG2a 655,360 <160 5,120

B8 7 IgG2a 655,360 <160 2,560

B9 8 IgG2a 655,360 <160 40,960

a+, Neutralization mixture contained normalrabbit serum as a comple-mentsourceatafinalconcentration of 10%.

b-,No normal rabbitserum waspresent.

cAll neutralizationmixturescontained normalrabbit serumasa

[image:3.612.66.305.559.687.2]

comple-mentsourceatafinalconcentrationofl1o.

TABLE 2. Isolation ofmarmutants with gB-specific antibodies

Selection Selecting Frequencyof marmutant Mutagena isolation of

expt antibody marmutantb designation

1 B1 BUdR 1 x 10-3 B1.1, B1.2

2 B2 1 x 10-7 B2.1, B2.2

3 B3 1 x 10-2 B3.1,B3.2

4 B4 8 x 10-7 B4.1,B4.2

5 B5 3 x 1O-7 B5.1

6 B5 BUdR 5 x 10-7 B5.2

7 B6 1 x 10-6 B6.1, B6.2

aBUdR,5-Bromo-2-deoxyuridine.

bFrequency = fraction of virus surviving neutralization x fraction of isolateswiththemarphenotype.

Isolation of mar mutants. The gB-specific monoclonal

antibodies were used to select aseriesof mar mutantswhich

wereresistant toneutralizationyetexpressed gB in infected

cells and incorporated this antigen into virion envelopes. The mar mutants were obtained by neutralizing plaque-purified wild-type strain KOS-321 virus with single

mono-clonalantibodiesin the presence ofcomplement. The virus

survivingneutralization (typically lessthan0.1%) was

pas-saged at low MOI and subjected to a second round of neutralization. Plaque-purified isolates ofthesurviving virus

were thentestedfor resistancetoneutralization in

quantita-tive

50%

endpoint plaque reduction assays. Anisolate was

designatedas amarmutantifthe monoclonalantibody titer against the mutant was at least 32-fold less than the titer when tested against the wild-type parent virus, KOS-321 (15). In most cases, even undiluted selecting monoclonal

antibodies failed to neutralize the corresponding mar mu-tants. marmutant-infected cellsexpressed gB as shownby specific radioimmunoprecipitation with a pool of the gB-reactive monoclonal antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (datanotshown).

Insevenindependentexperiments, sixgB-specific

mono-clonalantibodieswere usedto isolate12 mar mutantsfrom mutagenized or nonmutagenized virus stocks. The deriva-tion and frequency of isolation of these mutants is

summa-rized in Table 2. The frequency of isolation of the mar mutants varied from

10-2

to

10-7.

Operational antigenic map ofgB. The availability ofmar mutants and gB-specific monoclonal antibodies provided a means of analyzing the antigenic structure of this viral

glycoprotein. Each mar mutantshould express an antigeni-cally variant gB with a structural alteration that affects

reactivity with one or moremonoclonalantibodies. Thus, at least one epitope ongB has been alteredineach mutant. A

panel ofmar mutantswith different mutationscanbe usedto

discriminate between monoclonal antibodies reactive with different epitopes on the glycoprotein. As we have previ-ously shown with

gC-specific

monoclonal antibodies and mar C mutants, an operational antigenic map can be con-structed from the reactivity patterns ofmar mutants with monoclonal antibodies (23).Alterations indifferentepitopes

can be identified by unique patterns of reactivity, while

overlappingpatterns ofreactivitycanbe

grouped

togetherto defineantigenicsites composed ofmultipleepitopes (23).

Wetested thepanel ofgB-specific antibodiesforreactivity

with the mar mutantsin virusneutralization assays(Fig. 1).

Inaddition to the nine antibodiesagainstKOS-321produced

in this laboratory, we also tested seven produced by

Showalteret al. (33) againstHSV-1 strain 14012

(antibodies

3Sthrough61S). Eachantibody orgroupof antibodieswith

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G

MONOCLONAL ANTIBODY GROUPS

0 nr

IIi

IIIIIV

v

u MUTANT

C.0() (tJc1)n 1

P

C") CN L

_O

r- 00

o

racT-

(_

(n

CT

(LO C)

ma m co m co m vtC mVM coC1 C1 m cn

KOS321 _ -_

B1.1 *-_

B1.2 *

-B3.11

f

B3.2 * _-_

B2.1

2

B2.2

-

* * * * _ _

B5.1 __ * *

0

* * ___

B5.2

_ * * _ _ _

3 B4.1 __ ___ _ __ L _- - -_

B4.2 --

-B6.1

_

_

B6.2_

FIG. 1. Operationally defined epitopes of HSV-1 (KOS-321) gB. Individual monoclonal antibodiesweretested for neutralization of wild-type virus (KOS-321) and mar mutants in50% plaque reduction neutralization assays. The virusesweredesignated as resistant(0) orsensitive

(L)

toneutralization with eachantibody. The criterion for resistance was that thetiter of the antibody, when tested against the mutant, was at least 32-fold less than the titer against the wild-type virus.

aunique reactivity patterndefined adifferentepitope on gB.

Accordingly, a minimum of five nonoverlapping epitopes (designated I through V) were identified by the distinct verticalpatterns. With the exception of antibodies

represent-ing epitope V, for which mar mutants have not been

se-lected,alloftheantibodies produced against KOS-321could

be placed into groups with

corresponding

mar mutants,

designatedgroup IthroughgroupIV. Ofthe seven antibod-ies produced against strain14012, three(antibodies 3S,40S,

and 61S) had reactivity patterns similar to antibodies

pro-ducedagainst KOS-321. The remaining four antibodies

neu-tralized all of the mar mutants as

effectively

as wild-type virus and constitute antibody group V. Although the anti-bodies in this group have the same reactivity pattern and

defineatleast oneepitope, theyarenotnecessarily reactive with the same epitope and could define as many as four differentepitopes.

Identification of distinct antibody clonotypes byIsoelectric Focusing. To identify the number of different monoclonal

antibody

clonotypes in the above analysis, the gB-specific antibodies produced against KOS-321 were examined for their isoelectric focusing banding patterns (Fig. 2). This

analysis showed that the nine antibodies produced against KOS-321represent sevendistinctclonotypes. AntibodiesBi

andB3, whichcomprisegroupI,hadsimilarelectrophoretic patterns andprobably represent the sameclonotype. How-ever, these two antibodies were not derived from sister hybridoma clones since they wereisolated in two separate fusion experiments (Table 1). Of the five antibodies which

recognized thegroup II epitope, antibodies B7and B8 had

similar banding patterns (although derived from separate

fusions), while antibodies B2, B5, and B9 appeared to be

distinct and again were isolated from separate hybridoma fusion experiments (Table 1). Antibodies B4 and B6, in groupsIIIandIV,respectively,haduniquebanding

patterns

andweredifferentfrom the othergB-specific antibodies. Selection of mar mutants with multiple epitopechanges in

gB. Mutants withmultiple antigenic changes mightbealtered inotherphenotypes, suchastheirreactivitywith

polyclonal

antisera produced against wild-type HSV-1 (KOS-321) or suchas virus production athigh temperature. To examine these possibilities, mar mutants with multiple epitope changes in gB were isolated and studied.

A series of multiple mar mutants were constructed with two, three, and four epitope changes in gB. Since the

frequency of mutants with simultaneous changes in more than one epitope could be expected to be very low, the

epitope changes in gB were introduced sequentially. Two double mutants, mar B3/4.1 and mar B2/4.1, were first isolated by recombination after mixed infections with mu-tants mar B3.1 and mar B4.1, or mar B2.1 and mar B4.1.

B1

B3 B2 B5 B7 B8

B9

B4 B6

1+I

A.

JR

-I

-m.i.

l-I

FIG. 2. Isoelectric focusing ofgB-specific monoclonal antibod-ies. Antibodies were electrophoresed in a5% polyacrylamide gel (pH 4.0 to 9.0) at 2 mA for 18 h as described in Materials and Methods. The electrophoretically separated proteins were visual-izedbystainingwithCoomassiebrilliant blue. The anode(+)and cathode(-)areindicated.

4S

adM

;W

44mom

'Aqmvml.

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[image:4.612.58.287.70.271.2] [image:4.612.314.555.231.660.2]

Because HSV recombines actively, it was expected that somemultiplemarmutantrecombinantswouldbe produced. Progeny virus of the mixed infections was subjected to

neutralization with a mixture of the relevant monoclonal antibodies (B3 + B4 or B2 + B4) to enrich for recombinants with the double mar mutant phenotype. These antibody

mixtures neutralized the two parentalvirus mutants as well as wild-type virus recombinants, allowing only double mu-tants tosurvive selectionby neutralization. marB3/4.1 and mar B2/4.1 had the expected patterns of resistance to neutralization (Fig. 3); these mutants were resistant to

neutralizationby antibodies of the two appropriate antibody groups. Thereactivitiesofrepresentativesingle mutants are

also shown in Fig. 3 for comparison.

We then introduced mutations affecting epitopes

recog-nized by monoclonal antibodies B6 and B3 by means of sequential immunoselection against mar B2/4.1 progeny

virus. In this manner, we isolated the triple mutants mar

B2/416.3 and mar B2/4/6.4, which have alterations in the three epitopes definedby antibodies B2, B4, and B6. In an

independent selection, antibody B3 was used to isolate the

quadruple mutants mar B2141613.3 and marB2141613.4 from the triple mutant mar

B214/6.3.

The reactivity patterns of thesemutants toneutralization with thegB-specific

antibod-ies arealsoshown in Fig. 3. As expected, thetriplemutants wereresistanttoneutralization with thegB-specific antibod-ies representing three of the epitopes ongB, andthe

quad-ruplemutants were resistant to neutralization by all of the antibodies tested, representingthefourantibodygroups.

MONOCLONAL ANTIBODY

mar _____

MUTANT IV

-. CV) CN LD) cm(

B1.1

B3.1 @0

B2.1 _

B5.1 @0

B4.1 B6.1

B3/4.1 * *

B2/4.1 * * *

B2/4/6.3

B2/4/6.4 * * * *

B2/4/6/3.3 _ _

B2/4/6/3.4 * * _

R3B14.1

FIG. 3. Neutralizationpatternsofmutantswithmultipleepitope changesin gB. Mutantsweretested forsensitivitytoneutralization with the gB-specific antibodies

Bi

through B6 in 50% plaque reduction neutralization assays. The mutants were designated as resistant(0)orsensitive

(OI)

toneutralization as described inFig.1.

100

80-z 0

$ 60

N

I

40-L1

z

20-0

9 10 11 12 13 14 15

LOG2 ANTIBODY DILUTION

FIG. 4. Neutralization ofmultiple mar mutants with antiserum. Wild-type KOS-321 (0) and mar B2/41613.1 (A) were neutralized with twofold serial dilutions of mouse antiserum. The antiserum was produced by infecting mice with 103 PFU of live KOS-321 virus intraperitoneally. The mice were bled at 7 dayspostinfection, and sera were pooled from three mice for virus neutralization assays. Thepercent neutralization wascalculated from triplicate assays of thesurvivingvirus.

Reactivity ofmultiplemar mutants with antisera produced against wild-type HSV-1 (KOS-321). The effect of antigenic alterations present in multiple mar mutants on reactivity withapolyclonal antiserumproducedagainstwild-type virus

was tested. This would examine whether the multiple epitope alterations in gB significantly affected the overall antigenic structure of the virus envelope. Antisera against KOS-321 were produced by infecting groups ofmice with various doses of live virusranging from

102

to

106

PFU per

mouse. The mice were then bled at 3, 5, 7, and 10 days postinfection, and the

neutralizing

activity titers of thesera were determined. Sera frommice immunized with

103

PFU hadneutralizing titers of 1,024at7days postinfection. Mice bledatearlier timesorimmunized with lower doses ofvirus

produced verylow titers (<64). The day 7 antiserum from mice immunized with

103

PFUwasstrongly reactive with gB

as detectedby

radioimmunoprecipitation

(datanot shown). Weak reactivities with gC and gD were also noted. The observation that gB is the

major

inducer of immuno-precipitating antibodies during HSV infection agrees with

ourearlier

findings

(13). This antiserum was tested for its abilitytoneutralize

wild-type

KOS-321 andmarB214/613.3. Despite thefactthat themar mutantlacked four

epitopes

in

gB, and gB is a major target antigen for precipitating antibody, the quadruplemutantappearedtobeassensitive

toneutralization with this antiserumas

wild-type

virus(Fig. 4). Thus, thecombination offour different

epitope

changes

ingB didnot

significantly

affect

reactivity

witha

polyclonal

antiserumproduced againstwild-type

virus,

suggesting

that

these epitopes were not strongly immunodominant in the

totalantiviral antibody response.

marmutants withmultiplemutations exhibita tsphenotype.

Viruses with point mutations in the structural gene of

gB

havebeen identifiedwhichshow

temperature-dependent (ts)

defects in

processing

ofpgBtogB,in

plaque

formation,

and

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TABLE 3. Effects of temperature on plating efficiency and production of mar Bmutantsa

Platingefficiency Virus yield Virusmutant Temp Fraction of Fraction of

M~) PFU/ml 34Cvle PFU/ml 34'Cvalue

KOS-321 34 9.2 x 107 3.1 x 108

39 4.2 x 107 0.443 1.4 x 107 0.044

mar B1.1 34 9.0 x 107 1.2 x 108

39 2.2 x 107 0.250 1.5 x 106 0.012

marB2.1 34 1.7 x 108 9.5 x 107

39 7.9 x 107 0.465 1.3 x 106 0.014

marB3.1 34 6.4 x 107 3.5 x 108

39 3.8 x 107 0.568 3.4 x 107 0.097

marB4.1 34 3.9 x 108 2.9 x 108

39 1.4 x 108 0.367 5.6 x 106 0.019

marB5.1 34 2.0 x 107 2.9 x 107

39 8.5 x 106 0.436 5.3 x 105 0.018

marB6.1 34 4.4 x 107 2.3 x 108

39 2.2 x 107 0.506 7.2 x 106 0.031 marB2/4.1 34 8.8 x 107 5.6 x 107

39 2.5 x 104 <0.001 5.0 x 104 <0.001 marB3/4.1 34 2.0 x 107 2.1 x 108

39 8.5 x 106 0.425 4.6 x 107 0.139 marB2/4/6.3 34 7.4 x 107 1.1 x 108

39 4.5 x 104 <0.001 4.6x 104 <0.001 marB2/4/6/3.3 34 3.8 x 107 7.0 x 107

39 1.8 x 104 <0.001 6.0 x 104 <0.001

ts F13 34 9.0 x 106 3.0 x 108

39 1.5 x 102 <0.001 1.6 x 104 <0.001 R3marB4.1 34 1.4 x 108 2.3 x 107

39 5.0 x 107 0.357 1.7 x 106 0.074

aViruses were grown and titers were determined on Vero cells at37°C.

Plating efficiency is presented as the fraction ofplaques formed at 39°C compared with34°C. Virusyieldisdescribed asthe fraction of virus PFU producedat39°C compared with 34°C when titersaredeterminedat37°C.

invirus production (7). DNAsequencing ofmarmutationsin the gB gene has shown that antigenic variation can be conferred by single-base-pair substitutions (28; D. J.

Dorney, S. L. Highlander, M. Levine, and J. Glorioso, unpublished data). This finding raised the question of

whether antigenic alterations in gB can simultaneously

im-pair the ability of gB to function in virus replication in a

temperature-dependent fashion.To examine thispossibility,

marmutantsweretested forefficiency of plaque formationat 34 and 39°C, temperatures which are permissive and

nonpermissive

for virus

production

of othertsgBmutantsof

HSV-1 (KOS) (13, 22, 30). At the nonpermissive tempera-ture, wild-type KOS-321 showed an approximate 2-fold reduction in plating efficiency whereas the previously iso-lated KOS mutant, ts F13, showed a60,000-fold reduction

(Table3). tsF13carriesamutation in the structural gene for gB and is defective in the processing of pgB to the mature

form ofgB at 39°C (13; S. Highlander, unpublished data).

Noneofthesinglemar mutants showedts defects inplating efficiency. However, the double mutant marB2/4.1 and the

multi-mar mutants derived from it showed plating

effi-ciencies reducedto anextentcomparablewith the mutant,ts F13. In contrast, another double mar mutant, mar B3/4.1, was not ts for plating efficiency, demonstrating that the presenceof two marmutations was not in itself sufficient to produce tseffects.

Since these multiple mar mutants were ts for plating efficiency, we determined whether these mutants were also ts forvirus production. Virus production by the single mar mutants was notsignificantly different from that ofwild-type

virus at high temperature (Table 3). However, the multiple marmutants,which wereresistant to both antibodies B2 and B4, exhibited reduced virus production at 39°C, similar to the reduction observed for ts F13 (Table 3). Again, mar B3/4.1 did not show atemperature-dependent defect in virus production.

Effect of temperature on production of mature gB by mar mutants. All previously reported ts mutants mapping to gB

have an associated defect in the processing of the major precursorform of gB, carrying the mannose core sugars, to the mature fullyglycosylated form (13, 22, 30). We examined whether a similar defect in processing of pgB to gB also occurred in cells infected with the mar mutants. Radiola-beled viral proteins from cells infected at 34 or 39°C were

immunoprecipitated with a pool of gB-specific antibodies andanalyzedby sodiumdodecylsulfate-polyacrylamidegel electrophoresis (Fig.SA). Both the mature form of gB and its partiallyglycosylatedprecursor,pgB,were

immunoprecipit-ated from cells infected with wild-type KOS-321 at 34 or

39°C. However, mar B2.1 and mar B4.1 both exhibited

partialbutsignificant decreases in theexpression of mature

gB at 39°C; gB was produced by both mutants, but at

substantially lower amounts than by wild-type virus. In

addition, pgB overaccumulated relative towild-type virus. mar B5.1 had a similar phenotype (data not shown). This mutant was selected with monoclonal antibody B5, which

recognized the same epitope as antibody B2 (Fig. 1). Of theseviruses, marB2.1 appearedtobemoreaffected inthe

production ofmature gB. These findings were unexpected

since none of these mutants was ts for platingefficiency or

virus production. These observations suggest that apartial

defect in gB processing athigh temperature is not in itself sufficient to inactivate the glycoprotein function in virus

infectivity. In contrast, mar B3.1 was not affected in gB processing at 39°C, indicating that mar mutations do not

always affecttheintracellularprocessingofthe glycoprotein. However,therecombinantmarB3/4.1wasaffected in

proc-essing,and thus the defect in this double mutant waslikely

derived from the mar B4.1 parent and not the mar B3.1 parent. Insteadof the partial processing defect seen inthe

singlemutants marB4.1and mar B2. 1,the recombinantmar B2/4.1 (Fig. 5) and all ofthe multiple mar mutants derived fromthisrecombinant (datanotshown) producedno detect-able mature gB at 39°C. In addition, pgB was

immunopre-cipitated

inanoverabundance. Apparently, theintroduction of two mar mutations, each of which partially affected processing ofgB athigh temperature, greatly inhibited the processing of this molecule.

Previously,weshowed that mutant ts F13failed to present any form ofgB in infected cell surface membranes at the

nonpermissivetemperature(13).Accordingly,wecompared

the single mutants mar B2.1 and mar B4.1 with the double mutant marB2/4.1 for the ability to present gB on infected cells. Monoclonalantibody B3,whichrecognizesaseparate, unaltered epitope in these mutants, was used to detect the presence of gB on glutaraldehyde-fixed infected cells in

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As

KOS B2.1 B3.1 B4.1 B3:4.1 B2 4.1

34 39 34 39 34 39 34 39 34 39 34 39

pgB'

UUII

UIIUIII

B.

KOS

B 2 /4.1 R3 B4.1

34 39 34 39 34 39

_O

..

"

~

~

~~~~~~~

--gB

w-

w

gB

Sib 1I~~'I'

FIG. 5. Effect oftemperature on processing ofgB marmutants. HEL cells were infected at an MOI of 10 at either 34 or 39°C and radiolabeled with[35S]methioninefor4 to18 hpostinfection.gB wasimmunoprecipitatedfrominfected-cell lysates with a pool ofmonoclonal antibodies (B3, B4, B5, andB6). The immunoprecipitates were electrophoresedin a 7% sodium dodecyl sulfate-polyacrylamide gel and visualized byfluorography. The gel wasintentionallyoverexposed to detect small amounts of theimmunoprecipitatedprotein. The relative positions of thematuregB species and theprecursor, pgB, areindicated.

enzyme immunoassays (Fig. 6AandB). At34WC (Fig. 6A),

antibody B3 reacted equally well with cells infected with

wild-type

virus and with mutants mar B2.1, mar B4.1, and marB2/4.1,aswellaswithmar B6.1,whichhas nodefect in

w

al.1

0 z

m 0 co 2 0.4. z

w

<

gB processing at 39°C. No reactivity was detected against

mock-infected

cells. Cell surface expression of gB

in

cells

infectedat39°C(Fig. 6B)closely paralleledthe immunopre-cipitation data.Thatis,cellsinfectedwith marB2/4.1had no

0.4

0.3

0.2-0.1

-

0.4-0.3

0.2'

0.1-10 11 12 13 14 15 16 17

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LOG2 ANTIBODY DILUTION

FIG. 6. Effect oftemperatureonexpression of gBonsurface membranes of cells infected withmarmutants.HELcellswereinfectedat

anMOI of10,ormock-infected, andglutaraldehyde fixed at18 hpostinfection. The fixed cells weretestedfor reactivity with monoclonal antibody B3 by enzyme immunoassay as detailed in Materials and Methods. Each antibody dilution was tested in triplicate. Standard

deviationswerelessthan 0.03.

C.

340C

D.

i

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TABLE 4. Comparison of the ability of monoclonal antibodies reactive with epitope groupsIthroughIVtoimmunoprecipitategB produced by mar and ts gBmutantsof HSV-1

Immunoprecipitation by monoclonalantibodiesa

B3(groupI) B5(groupII) B4(group III) B6 (groupIV)

HSV-1

34°C 39°C 34°C 39°C 34°C 39°C 34°C 39°C

pgBb gBb pgB gB pgB gB pgB gB pgB gB pgB gB pgB gB pgB gB

KOS-321 + + + + + + + + + + + + + + + +

marB3.1 - - - - + + + + + + + + + + + +

marB6.1 + + + + + + + + + + + + + + +

marB5.1c + + + + - - - - + + + - + + + +

marB4.1c + + + ± + + - - + + - - + + + *

tsB5(HFEM) + + + NP + + - NP + + - NP - - - NP

tsF13 (KOS) + + + NP + + - NP + + - NP + + + NP

tsJ12 (KOS) + + + NP + + - NP + + - NP + + + NP

aVerocells were infected at 34 and 39°Catan MOI of 5, radiolabeled with[35S]methionine5hafterinfection, extracted after 15 h with 1% NonidetP-40, and

immunoprecipitated with monoclonal antibody. Monoclonal antibodies all react with gB and represent distinct epitope specificities. The precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. +, Immunoprecipitation; -, lack of immunoprecipitation; +, weak immunoprecipitation. NP, Mature gB not produced at 39°C.

bMaturegB(Mr = 126K) and its major precursorpgB (Mr = 107K).

cMature gB produced in reducedamounts at39°C.

detectablegB on the cellsurface,whereas cellsinfectedwith marB2.1ormarB4.1 presented gB onthecell surface,but

inamounts substantially lessthanwild-type virus.marB2.1

produced less surface membrane gB than mar B4.1, while the control mutant infection, with marB6.1, wassimilar to

the wild-type virusinfectionin thecell surfaceproduction of

gB.

Comparison of immunoreactivity of gB produced by mar mutants andtsgB mutantsof HSV-1. Asdescribedabove, the

double marmutantmarB2/4.1failed toproduce maturegB at high temperature. This abnormal processing phenotype

hasalsobeenreported forothertsgBmutantsselectedforts virus production, rather than by resistance to neutralizing antibodies (13, 22, 30). Thisobservation raised the possibil-itythat thesetsgBmutantsalsoproduceaform of gB which lacks the group II and group III epitopes. To examine this question, group II (B5) and group III (B4) monoclonal antibodiesweretestedfor theirabilitytoimmunoprecipitate

pgB and gB (at 34°C) and pgB (at 39°C) from lysates of infections with each ofthree HSV-1 ts gB mutants, ts B5 (HFEM), ts F13 (KOS), and ts J12 (KOS) (Table 4).

Im-munoprecipitations with group (B3) and group IV (B6)

antibodies werealsocarried outforcomparative purposes. EachofthetsgBmutantsreacted with group II and group IIIantibodies inasimilar fashion. At34°C, both pgBandgB

wereimmunoprecipitated,demonstratingthe presenceofthe groupIIand group IIIepitopesontheseproteins. Strikingly,

the pgB produced by these three mutants at 39°C was not

precipitated by these antibodies. That is, the group II and group III epitopes were not present at high temperature. This effect of temperature is limitedtothe group II and III

epitopes;the group Iand IVepitopesappeartobeunaffected

by high temperature. Monoclonal antibody B3 ofgroup I

immunoprecipitated the gB polypeptides produced at low

and high temperatures from all three ts mutant infections.

AntibodyB6of groupIValsoprecipitated the gB molecules produced in low- andhigh-temperature infections with mu-tants tsF13 and ts J12. The B6 antibody failed to

immuno-precipitate any form of gB produced in ts B5 infections,

indicatingthat thismutantlacks the B6epitopeinadditionto itstsphenotype. Theseobservationssuggest thatthegenetic alteration leadingtothe tsphenotype of these mutantsalso results in alterationof thegBpolypeptide formedatelevated

temperature,whichaffects recognition ofthe groupIIandIII epitopes bythe corresponding monoclonal antibodies.

Also presented in Table 4 are immunoprecipitation data from infectionswithrepresentativemarmutants.Thesedata

bear on thethermostability ofthegBepitopes just described.

That is, epitope thermostability is related to the altered

processing and epitope structure of gB forms produced at

39°Crather thantoepitope denaturationathightemperature.

AntibodyB3 (groupI)immunoprecipitated pgBandgB from

mar B5.1, mar B4.1, and mar B6.1 infections at

both

temperatures. ThemarB3.1mutationaltered theB3 epitope inthatantibodyB3 failedtoimmunoprecipitate anyform

of

gB produced by mar B3.1 infections. While the mar B6.1

mutation rendered themutantvirions resistantto neutraliza-tionbyantibody B6(groupIV),gBwasweakly

precipitated

at eithertemperature from mar B6.1-infected cell extracts. Asexpected, B6immunoprecipitated pgBand

gB

(at34and 39°C) frommarB3.1-,marB5.1-,andmarB4.1-infected cell

extracts.These dataindicatethattheB3andB6epitopesare notthermolabile.

Both epitopes II and III are made thermolabile to immu-noprecipitation by the mar B4.1 mutation. Antibodies

of

group II (B5) and group III (B4) were unable to precipitate

pgB produced at 39°C in mar B4.1 infections. In contrast, pgBproduced at39°C inmarB5.1infections appears to be thermostable in that it is precipitated by antibody B4 of

group III. That is, epitope III of this protein can still be recognizedby the corresponding antibody athigh tempera-ture.ThemarB4.1gBphenotype mimicksthegBphenotype ofthetsmutantsdescribed above. Itshouldbe

remembered

that theseantigenicvariants behaveassinglemar mutantsin

virusneutralization assays. Takentogether, epitopes II and

III appeartodependonconformation for theirintegrity and

may reside in a domain susceptible to

temperature-depen-dent alteration.

Characterization ofrevertants of ts mar B2/4.1. If the ts phenotype isaconsequence of themutations producingthe marphenotype,itwould beexpectedthatreversionofthets defect would correlate with coreversion to reactivity with

monoclonalantibodyB2 orB4. Toexamine thispossibility, potentialrevertants of the ts phenotype were

generated

by

two passages at 39°C. Twenty-four isolates were plaque purifiedand testedforreactivitywithmonoclonal

antibodies

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B2 and B4 in animmunoperoxidase plaque assay (16). All 24 isolates regained reactivity with antibody B2 but remained unreactive withantibody B4, asshown with a representative isolate, R3 mar B4.1 (Fig. 7). Thedoublemutant mar B2/4.1 failed to react witheitherantibody (data notshown). When tested, R3 mar B4.1wasfound to have lostthe ts phenotype for both plating efficiency and virusproduction (Table 3).

R3 mar B4.1 was also tested for quantitative reactivity withmonoclonal antibodiesB2 and B4 in 50% plaque reduc-tion assays. R3 mar B4.1remainedresistant to antibody B4, but regained sensitivity to neutralization by antibody B2. The parental double-mar mutantremained resistant to

neu-tralization by both antibodies (Fig. 3). These data indicate that in revertantsrepresented byR3 mar B4.1, reversionof the tsphenotypewasassociatedwithre-establishmentofthe

epitopedefined by antibody B2.

Since the double mutantmar B2/4.1exhibitedtsdefectsin theproductionof gB,R3 mar B4.1 wasfurther characterized forprocessingof pgB to thematureform ofgB at39°C. As

detected by immunoprecipitation, R3 mar B4.1 partially

regainedthe ability toprocesspgB to gB at levelssimilarto the single mar mutant parent mar B4.1, but less than

wild-typevirus (Fig. SB).This result was expected since the

revertant retainedtheB4 mutation which confers apartialts defect in gBprocessing (Fig. 5A).

Inadditiontoprocessingof gB, therevertantR3 marB4.1 also partially regained cell surface expression of the glyco-proteinasdetermined byquantitative enzyme immunoassay

of fixed infected cells withantibody B3 (Fig. 6C and D). At

34°C, R3marB4.1-infectedcellsexpressedcellsurface gBin

amountsequal towild-type virus or mar B4.1. However,at 39°C, R3 mar B4.1 retained expression of cell surface gB, but inintermediateamounts similar to the amountexpressed

by the single mutant mar B4.1. Thus, concomitant with

reversion of the ts phenotype, R3 mar B4.1 regained reac-tivity withantibody B2 butnot B4 andregainedthe

process-ing and cell surface expression characteristics of the mar B4.1 parent.

DISCUSSION

The purposes of our studies of HSV-1 (KOS-321) gB were (i) to define the antigenic structure ofthis antigenand(ii) to

determine whetherantigenic variation in gB canaffect virus production in cell culture. To carry out these analyses, monoclonal antibodies were used to select a series of vari-ants, marmutants, altered intheantigenicstructureof gB.A panel ofgB-reactive monoclonal antibodies representing six distinct clonotypeswas used to selectvariants. Inthis way, we hoped to obtain mar mutants which sustained mutations

in different sites within theregion ofthe gB structural gene that encodes the external glycoprotein domain. Virus-neutralizing monoclonal antibodies were employed in mar mutant selectionsbecause oftherelative ease with which the mutants are isolated and because loss inepitope expression

can be quantitated by changes in antibody titer against the mutant compared with the titer against the wild-type virus.

Although our minimum criterion was a 32-fold reduction in

relative antibody titer (15), most selecting antibodies

com-pletely failed to neutralize the corresponding mar mutants. Thus, we were assured of analyzing mutants which were altered in an amino acid critical to recognition of gB by the

selecting monoclonal antibody.

Many of thegB-specificmonoclonal antibodiesrecognized epitopeswhich are present in both virusserotypes, suggest-ing that the antigenic makeup of HSV-1 and HSV-2 gB is

mAb B2

mAb 84

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IMMLUNOPEROXIDASE CRYSTAL VIOLET

FIG. 7. Reactivityof arevertantvirus with monoclonal antibod-ies in the immunoperoxidase black plaque assay. The ts revertant R3 mar B4.1 was tested for its ability to react with either of two monoclonal antibodies (B2 and B4) in an immunoperoxidase assay. Virus was plated on Vero cell monolayers and glutaraldehyde fixed when plaques developed. Duplicate plates were then incubated with monoclonal antibody B2 or B4, followed by incubation with goat anti-mouse horseradish peroxidase-conjugated antibody. After ad-dition of substrate (4-chloro-1-naphthol), plaques showing black precipitate were scored as positive, and plates were counterstained with crystal violet.

highly conserved. B4 was the only antibody that neutralized HSV without complement. These observations may be con-trasted with ourstudies of gC-reactive monoclonal antibod-ies. Of more than 30 hybridomas producinganti-gC-1 (23) or anti-gC-2 monoclonal antibodies (J. C. Glorioso and M. Levine, unpublished data), none cross-reacted with gC of the other virus serotype. Moreover, none of the gC antibod-ies neutralized virus in acomplement-independent manner. Based on the analysis of the reactivity patterns of the gB mar mutants with a panel of 16different gB-reactive mono-clonal antibodies, four distinct epitopes were identified. The remaining antibodies, which neutralized all single-mar mu-tants, comprised a separate grouprepresenting at least one additional epitope. One epitope, group II, was somewhat more immunodominant than the rest. Five antibodies were shown torecognize thisepitope, includingthoseproducedin adifferent laboratory and against a different strain of HSV-1 (33). In contrast to our findings with gC mar mutants (23), none of the epitopes in gB gave overlapping reactivity patterns, suggesting that the antibodies bind to different parts of the gBmolecule. This suggestion iscompatiblewith the finding that multiple mar mutations could be readily introduced, one at a time, into the gB structural gene by recombination and sequential antibody selection. The addi-tion of each new mutation had a simple additive effect, resulting in resistance to neutralization only by the corre-spondingselecting antibody. No unexpected multiple resist-ancephenotypes were found. Monoclonal antibody compe-tition studies are in progress to determine whether the

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antibodies indeed bind to topographically distinct antigenic

domains. Giventhe size of the external domain ofgB (-700 amino acids), it is likely that this molecule is more

antigen-ically complex than our analyses have shown. With the

isolation of additional mar mutants selected with new

anti-bodies,amorecompleteandpossiblymorecomplex picture oftheantigenic structureofgB may emerge.

Oneapproachtodetermine whetheraparticularviralgene product is essential for virus growth is to search for ts mutants. Such mutants altered in gB have now been

de-scribed byanumberoflaboratories(7, 13, 22, 30), and some

ofthese mutations have been shown to result from single amino acid substitutions in the external domain of this

antigen (7). As our laboratory (Dorney et al., unpublished data) and others (28) haveshown that marmutations ingB also can result from single amino acid changes, we were

intrigued by thepossibility that marmutations ingB might affect otheraspects of the biology ofthisprotein, including alterations in its processing and function at high tempera-ture. While none of the single marmutants wasaffected in

virusgrowth,adoublemarmutant, marB2/4.1, anditstriple and quadruple mar mutant derivatives all showed reduced plating efficiency and virusyieldat39°Cto adegree

compa-rabletothe other ts mutantsaffectedin gB whichwere not

selected with antibodies.

All ts mutants ofgB are defective in processing of the

major precursor ofgB to the mature gB form (13, 22, 30). Indeed, all of these mutants accumulate a pgB species of

similar molecularsize (13, 22, 30), which led us toexamine both single and multiplemar mutantsfordefectsin

process-ing of gB at high temperature. Several mar mutants, mar

B2.1, marB5.1, and marB4.1, were

partially

defective for processing of pgB to gB at 39°C, despite the finding that these mutants undergo productive infection at elevated temperature. Recombinants

carrying

two ofthese mar

mu-tationswerehighly defective forthis

processing

step,aswell asbeing defective for theproduction of activevirus

particles

(39°C). Furthermore, at hightemperature the HSV-1 ts gB

mutants, ts F13, ts B5, and ts J12, all accumulated pgB which wassimilar in size to that accumulated bythedouble mar mutant mar B2/4.1. Revertants ofthe ts phenotype of

marB2/4.1regainedthe B2 but not the B4epitope,

suggest-ing that the mar mutations, rather than a second-site ts

mutation, were responsible for the altered processing and temperature sensitivity. The combination oftwomutational changes in the doublemarB2/4.1mutantmoleculemay have

altered the

folding

ofthe precursormoleculemoreseverely

at the nonpermissive temperature, resulting in complete

arrest ofGolgi processing and transport to the cell surface, while atthe sametime producing apgBform which fails to supportvirusproduction.Thesefindingsshow thatantigenic variation in gB can result in changes not only in epitope

configuration, but perhaps moreimportantly, ininactivation ofthefunction of thisantigen invirus replication.

One of severalexplanations could account forthe failure

to produce active double mar B2/4.1 mutant virus at high

temperature. These include failure to produce enveloped particles, production of enveloped particles lacking gB or

pgB, or production of enveloped particles containing nonfunctional pgB. The last ofthese seems most likely for several reasons. First, the underglycosylated precursor

form, pgB, is found in virionenvelopes (34) and may be as

functionally active as the maturegB species. Second, sev-eral studies have shown that monensin or ammonium chlo-ride treatmentof HSV-infected cells results in thedisruption

of Golgi function and the addition of complex

oligosac-charides to the HSV glycoproteins. Although under these

conditions the yield of progeny virus is greatly reduced, these particles are nonetheless infectious (17, 19). Appar-ently, the failure to add terminal sugar residues tothe gB polypeptideisnotsufficienttoinactivate the function of this molecule. Third,revertants of themutant ts B5, affected in

gB, regain the ability to form plaques and produce active

virus at high temperature despite reduced formation of

mature gB (14). Revertants ofmarB2/4.1 showeda similar

phenotype. These data further suggest that processing of pgB togB is not coupledwith ts virus production.

Regard-less ofthe mechanism underlying the ts phenotype ofmar

B2/4.1, processing of pgB togBappearstobe linkedto,but notstrictly correlated with,the

expression

ofafunctionalgB

molecule.

Onequestion arising fromthese studies iswhetherts mar mutantscarrymutations whichmaptothe sameregionas ts

gBmutantsselectedonthebasis oftsgrowth. Althoughdata are not yet availableto answer this

question satisfactorily,

we have determinedbymarkerrescueexperimentsthatthe marmutationsresultingin thetsphenotype of gB

physically

maptothe samegeneral region ofthegB structural geneas the other ts gB mutations

(Highlander, unpublished data).

The pgB form produced by the ts gB mutants at 34°C is

immunoprecipitable by group I (B3), group II (B5), and groupIII(B4)

antibodies, demonstrating

thatthese

epitopes

are present in these strains. The HSV-1 HFEM strain is

apparently a natural

antigenic

variant since the group IV

(B6) antibody failed to immunoprecipitate the gB of this

virus. At 39°C, the B3 antibody immunoprecipitated pgB from all threetsgB mutants and B6reacted with pgB ofts F13and ts J12. In contrast,antibodies ofgroup II

(B5)

and

group III (B4) failed to immunoprecipitate pgB (made at

39°C)

despite

thefact that thesets mutants were notselected with monoclonal antibodies, but rather on the basis of ts virus production. Therefore, group II and III antibodies recognizeadomain ofgBwhich is alteredat39°Cin several virus strains while other antigenic domains in the same molecule remain intact. Thiswasillustratedbythecontinued reactivityofpgBat39°Cwith group I and group IV

antibod-ies. Theepitopes

recognized by

group II and III antibodies

appear to lie in a thermolabile

portion

ofthe gB molecule

critical forproper

gB

processing

and virus

replication.

The

finding

that the group III

antibody recognized

an

epitope

conserved in thetwoserotypes and neutralizedvirus in the absence of

complement

also suggests that this domain is criticaltovirus

infectivity.

The

thermostability

ofthe group I and group IV epitopes suggests either that they may be linear determinantsorthat thesemarmutationsdonotaffect

portions

of the molecule which lead to

temperature-dependent alterations of theseepitopes.

Inaddition to the nature of the tsphenotype of

multiple

marB mutants, several other issuesarise fromourstudies of gB. Whilefurther

analysis

isrequiredtodetermine whether thefivegB

epitopes

identifiedinthis

study

are

representative

of the

major

antigenic site(s)

for this

antigen,

our data do allow us to assess whether these

changes

are

significant

in

terms of immune recognition of the variant viruses by polyclonal antibodies.

First, the

antigenic

complexity of the HSV envelope in itself suggests that smallchangesintheantigenicstructureof the virus will not dramatically affect immune control of infection. As demonstrated in the presentstudy,alteration of four distinct epitopes ofgB had no measurable effect on

susceptibility to neutralization by polyclonal antisera di-rected against the wild-type virus. Second, particular

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genic variants may be selected against in natural infections. Based on the results with mar B2/4.1, certain immune-selected mutations may result in an inactive form of this glycoprotein in the natural host. Thus, certain regions of the gB structural gene are likely to be conserved in natural infections. This hypothesis is supported by the following observations: (i) preliminary examination of 40 primary virus isolates from humans has revealed that the group III (B4) epitope is present in all strains, whereas other epitopes of gB are highly variant and double mar B mutants have not been detected (G. Toth and J. Glorioso, unpublished data); and (ii) the ability of mar B2/4.1 to cause encephalitis in mice after intracranial inoculation is greatly inhibited as a result of these mutations (21). It is nevertheless clear that antigenic variation in the HSV glycoproteins is common (11, 29). Studies of antigenic variation in influenza virus have identi-fied mutations responsible for widespreaddisease outbreaks, suggesting that antigenic variation driven by antibody selec-tion can be epidemiologically significant(38). It isunclear at this time whether antigenic variation of HSV can lead to changes in the immunobiology of natural infections.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants AI-16216, AI-17900, AI-18228, and RR00200 from the National Institutes of Health. S.D.M. was supported by a Horace Rackham Predoctoral Fellowship from the University of Michigan, and both S.D.M. and S.L.H. received support from the University of Mich-igan Graduate Program in Cellular and Molecular Biology (Public Health Service Training Grant 2T32GM07315). We also acknowl-edge the support of T.C.H. by the Dental Research Institute Associate Program from the Office of Vice President for Research. We thank Elizabeth Smileyfor herexcellent technical work with monoclonal antibodies.

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