Isolation of a nucleocapsid polypeptide of herpes simplex virus types 1 and 2 possessing immunologically type-specific and cross-reactive determinants.

0022-538X/79/01-0034/09$02.00/0

Isolation

of

a

Nucleocapsid Polypeptide

of

Herpes

Simplex

Virus

Types

1

and 2

Possessing Immunologically

Type-Specific

and

Cross-Reactive

Determinants

CONRAD J. HEILMAN,

JR.,`*

MARTIN ZWEIG,' JOHN R. STEPHENSON,2 ANDBERGE HAMPAR3

ViralOncologyProgram,FrederickCancer ResearchCenter,Frederick, Maryland217011;Laboratory of CellularandMolecular Biology,National CancerInstitute,Frederick Cancer ResearchCenter, Frederick, Maryland 217012;andLaboratory ofMolecularVirology,National CancerInstitute,Frederick Cancer

ResearchCenter, Frederick, Maryland217013

Received for publication 21 August 1978

A polypeptide (p40) of approximately 40,000 molecular weight was isolated

from herpes simplex virus type 1 and 2 nucleocapsids by gel filtration and ion

exchangechromatography. This protein appearstobe the same as protein 22a

describedpreviously (Gibson and Roizman, J. Virol. 10:1044-1052, 1972).

Com-petition immunoassayswere developed by using purified p40 and antisera

pre-pared in guinea pigs. The assays indicated that the p40's from herpes simplex

virustypes1and 2possessbothtype-specific and cross-reactive antigenic

deter-minants.Antibodiestothe p40cross-reactive determinant reacted withantigens

in simian herpes virus

SA8-infected

cells, but not with antigens induced by

pseudorabies virus. Preliminary results indicated thataradioimmunoprecipitation

test can be used to detect type-specific herpes simplex virus p40 antibodies in

humansera.

Herpes

simplex virus

types 1

(HSV-1) and

2

(HSV-2)

are

large

complex

DNA-containing

vi-ruses which have been shown to

induce the

synthesis

of at

least

50 new

polypeptides in

productively

infected cells

(5, 13). Some

of

the

antigens

induced

by

HSV-1

and HSV-2

have

been shown

to possess cross-reactive

determi-nants

(which

reactwith antiserum

against

either

HSV-1

or

HSV-2), whereas other antigens

pos-sess

type-specific

determinants

(which

react

only

with antiserum

against

the

homologous

virus type) (4, 15).

Several HSV-induced

polypeptides

have

been

purified

and

immunologically

characterized

by

using

monospecific

antisera and

a

variety

of

immunological

techniques (for

a

review,

see

ref-erence

9). These include the

early

nonstructural

polypeptides

VP 175

(molecular weight,

175,000)

and VP134, the

envelope glycoprotein

VP 123,

and

the

nucleocapsid

polypeptide

VP 154.

The

nucleocapsids

ofHSV-1 and HSV-2

con-tain six

principal

polypeptides ranging

in

molec-ular

weight

from

approximately

12,000 to

154,000 (2; G. H.

Cohen,

W. C.

Lawrence,

M.

Ponce DeLeon, S.

Vernon,

and H.

Diggelman,

Abstr. Annu. Meet. Am. Soc. Microbiol. 1978,

S42,

p. 219; and see

below).

The

nucleocapsid

polypeptide

VP 154has been

reported

topossess

cross-reactive

antigenic

determinants (9, 14).

Thestudies described here

concern

theantigenic

34

properties of another

nucleocapsid

polypeptide

of HSV-1

and HSV-2 withamolecular

weight

of

approximately

40,000

(p40).

In

preliminary

stud-ies, p40

was

found

tobe among the most

soluble

of the

major

nucleocapsid proteins

and also the

most

amenable

to

'25I-labeling

at

high specific

activity without

significant degradation

or

ag-gregation.

Because these

properties

are

desirable

in

immunoassays,

we

selected

p40 for

our

initial

characterization studies.

Using the

competition

immunoassay

technique

with

purified

p40's

from

HSV-1

and

HSV-2

and

antisera

prepared

in

guinea

pigs,

we

found

that this

polypeptide

pos-sesses

both

type-specific and cross-reactive

an-tigenic

determinants.

MATERIALS AND METHODS

Cellsandviruses.Verocellsweregrown inroller

bottlesorflasks inEagle minimal essential medium supplementedwith10% heat-inactivatedfetal calf

se-rum,penicillin,and streptomycin. MAL (type 1) and

MS (type2) were theprincipal HSV strains employed. Other herpesvirus strains included 14-012 (HSV-1), Miyama (HSV-1), 333 (HSV-2), Savage (HSV-2), pseudorabiesvirus(PRV),and simianherpesvirusSA8 (8).The Verocellswereinfected at amultiplicity of infection of 1 to 5, based on infectious virus titers. Afteradsorptionfor 1 h at 37°C, fresh medium was added, and the cells were maintained at 37°C until maximumcytopathic effectswere observed (18 to 36 hpostinfection). Thecellswere washedwith cold PBS

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VOL. 29, 1979

(0.14NaCl,0.02Msodiumphosphatebuffer, pH7.4)

and collectedby centHfugation (1,500xgfor 5 min).

Cellpelletswerestoredat-70°C untilused.

Purification ofnucleocapsid. Cells grown in

roller bottles were infected with either HSV-1 MAL orHSV-2 MS(multiplicityofinfection,5) asdescribed above andharvested at 24 h postinfection. Thecells

weresuspended in 0.01 MNaCl-0.01 M Tris-hydro-chloride-1.5 mMMgCl2, pH 7.5(bufferA) and incu-bated at0°Cfor 10min. The cells weredisruptedin a Douncehomogenizer in the presence of 1%Nonidet P-40 at0WC,and the nuclei were sedimentedby

cen-trifugation (600 xgfor 5 min) and resuspended in

bufferA.Nuclearlysateswereprepared byaddition of

sodiumdeoxycholateto afinal concentration of0.5%,

followedby sonic treatment. After incubation of

ly-satesfor 30min at37°Cin the presence of 50

Ag

of DNase perml,urea wasadded to afinal concentration of 0.5 M, and debriswasremovedbycentrifugationat

8,000 rpm for 5 min in a Sorvall SS34 rotor. The

clarified lysatewaslayeredonto a6-mlcushion of 35%

(wt/vol)sucroseinPBScontaining1mMEDTA and

was centrifuged at 23,000 rpm for 1 h at4°C in a

Spinco SW27rotor(12). Theresulting pelletwas

re-suspendedinPBS, subjectedtosonictreatment,and

layeredontoa33-ml10 to40% (wt/wt)sucrose

gra-dientpreparedinPBSoverlayinga3-ml60%(wt/wt)

sucrose shelf. Centrifugation was performed in a

Spinco SW27rotorfor 60minat20,000rpmat

8°C

(2). Two prominent

nucleocapsid-containing

bands were formed near the middle of the tube andwere collected from the bottom of the tube. Thecontents of the bandswere

negatively

stained with

phospho-tungsticacid, examined by electron

microscopy,

and

foundtobefreeof

contaminating

material. Both bands

werepooled,diluted withPBS,sedimentedat23,000

rpm for 1 h at

4°C

in a

Spinco

SW27 rotor, and

suspendedinwater.

SDS-PAGE.

Sodium

dodecyl

sulfate-polyacryl-amide gel

electrophoresis

(SDS-PAGE)

was

con-ducted inaBio-Rad model220slab

gel

apparatusfor 16h at 30 V byusingadiscontinuousbuffer system

containingalinear 5to20%

(wt/vol)

polyacrylamide

gradient (7). Theprotein bands werefixed in 12.5%

(wt/vol) trichloroacetic acid for1handstained with

0.05%Coomassie brilliant blue R250 in 12.5%

(wt/vol)

trichloroacetic acid for4to16

h,

andexcess

dye

was removed by washing in5% methanol and 7% acetic acid. For

analysis

of

l"I-labeled

proteins,

the

gels

were

driedontofilter paper withaBio-Rad model224

gel

slab dryer, and

autoradiographs

were

prepared

on

KodakX-Omat R film. Absorbance of bands in stained

gelsand

autoradiographs

wasmeasured

by

asoftlazer scanning densitometer

(Biomed

Instruments,

Inc.,

Chicago,Ill.).

Isolation ofviralproteins.Purified

nucleocapsids

(totalprotein,10mg)were

suspended

in 0.5ml of0.01

M dithiothreitol (DTT)-2 mM EDTA-0.05 M

Tris-hydrochloride (pH 8.5) buffer

containing

8M

guani-dinehydrochlorideandappliedto anagaroseA-5

100-to200-mesh(Bio-RadProducts)

gel

filtration column (0.5by90cm)in 0.02 M sodiumphosphate

buffer, pH

6.5,containing6Mguanidine

hydrochloride

and0.01

MDTT(1).Column fractions

containing

a

major peak

at amolecularweightof about40,000asdetermined

NUCLEOCAPSID p40 FROM HSV 35

bySDS-PAGEwerepooled and

exhaustively

dialyzed

against 200 volumes of a solution containing 0.01

M N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic

acid, pH 6.5, 1.0 mM EDTA, and 0.1% Triton X-100 (bufferB) andappliedto aphosphocellulose column (1.0by 5.0 cm;Whatman pll;H.Reeve Angel & Co., Inc., Clifton, N.J.) previously equilibrated with the same buffer. The column was washed with 50 ml of bufferB,and bound proteins were eluted with a 100-mllineargradient (0to 1.0 MNaCl).Fractions eluting at 0.64 to 0.82 MNaCland containing a single protein of about 40,000 molecular weight were pooled, divided

intoaliquots,andstored at -700C.

Antiserapreparation. HSV-1 MAL and HSV-2

MSnucleocapsidproteins (approximately 200ltg)were

separatedbySDS-PAGEon slab gelsand stained with

Coomassie brilliantblue. The 40,000-molecular-weight

bands were cut out,minced in 2 ml of PBS with a7-ml

Dounce homogenizer, and subsequently emulsified

with an equal volume of Freund adjuvant. The antigen

preparations (2ml)were injected into Hartley guinea

pigs intramuscularly. The animals were immunied

threetimesatweekly intervals, and sera were obtained 10 days after the last injection. Complete adjuvant wasused in the first injection, and incomplete adju-vant was used in the two subsequent immunizations.

Immunoassays. Viral proteinswere labeled with

'25Iathigh specificactivity(100,uCi/,ug) by the

chlor-amine T procedure (3) andseparatedfromfreeiodine

byplO (Bio-Rad Products) column chromatography.

Duringiodination, however, it was necessary to reduce

the chloramine T concentration to 3.6 ug/ml to

pre-ventdegradationof the protein. Immunoprecipitation

assays were done byincubating

l"I-labeled

p40(10,000 cpm) with serialtwofolddilutions of antisera.

Com-petitionimmunoassayswereperformedby testing

un-labeled viral antigens at serial twofold dilutions for

ability to compete with

l"I-labeled

p4O for binding

limited amountsof antiserum. Antisera in the

com-petitionimmunoassays were employed at adilution

(1:400 to 1:5,000) which precipitated about 35% of the

"SI-labeled

p40.Antiserum andunlabeled competing

antigenwereincubatedfor 1 h at37°C, followedby

addition of 10,000 cpm of

'25I-labeled

p40. Reaction mixtures contained 0.01 MTris-hydrochloride,pH 7.8, 1.0 mMEDTA,0.4% TritonX-100, 1% bovine serum

albumin, 0.05 M

NaCL

and0.05% NaN3 in a total

volume of 0.2 ml. The antigen-antiserum mixtures wereincubated for 3 h at37°Cand 18 h at4°C;20Ml

of a second antibody (goat anti-guinea pig

immuno-globulin G;Office of Resources andLogistics, National

CancerInstitute,Bethesda, Md.)wasadded,and in-cubationwascontinued for1h at37°Cand 3 h at4°C.

After the addition of 0.04 ml of 10 mM

Tris-hydro-chloride (pH 7.8)-10 mM NaCl-0.1% Triton X-100, samples were centrifuged for 15 min at 2,500 rpm, supernatants wereaspirated, and radioactivity in the

precipitates was measured in a Searle model 1285

gammacounter.

RESULTS

Isolation and

immunoprecipitation

of

HSV-1 and HSV-2

nucleocapsid

p40's.

The

p40's

of

HSV-1

and HSV-2

nucleocapsids

were

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36

HEILMAN ET AL.

isolated

by

gel filtration and ion exchange

chro-matography.

After

labeling

at

high specific

ac-tivity

with

1"I,

their purity was

determined

by

SDS-PAGE

analysis.

The

nucleocapsids

of

HSV-2

(Fig.

1)

and

HSV-1

(data not shown)

consist of

six well-defined major proteins and

about six minor

proteins, with

molecular

weights

ranging

from 155,000

(p155)

to

12,000

(p12). For

clarity,

only

the

major

components are

identified

in

Fig. 1. The

12'I-labeled

p40

of

HSV-2 migrated

as a

single

homogenous

band of

radioactivity

at

a

molecular

weight of about 40,000, which

cor-responded to a

Coomassie

brfilliant blue-stained

protein

band

observed

after

SDS-PAGE

analy-sis of

unlabeled

nucleocapsid preparations.

The

corresponding

protein of

HSV-1

was

slightly

lower in

molecular weight (data

not

shown).

Immunoprecipitation

tests were

performed

by

using

the

'25I-labeled

p40

proteins of HSV-1 and

HSV-2 and antisera

prepared in guinea pigs. As

shown in

Fig.

2, the

homologous

p40

was

precip-itated with a

higher

dilution of antiserum than

was the

heterologous

p40.

The

difference

in

an-tiserum dilution for

precipitation

of

homologous

versus

heterologous

p40

was

greater with the

anti-HSV-2

serum

than with the

anti-HSV-1

serum.

One

possible

model accounting

for

this

phenomenon is that both antisera contained

two

classes of

antibodies, one which reacted with

type-specific determinants and

a

second which

reacted with

cross-reactive determinants. The

ratio of each class of

antibody

in any

oneserum

preparation could vary.

Support

for the

proposal

that

these sera

contained two classes of

antibod-ies

is provided in the

immunoassays

described

below.

Homologous

competition

immunoassays

for p40

in

fast- and

slow-sedimenting

nu-cleocapsids.

It has been

reported

that

HSV-infected cell

nuclei

contain two

species of

nu-cleocapsids, designated

A

and B

(2).

These

stud-ies showed that the B

nucleocapsids

contain

10

times the amount of DNA and sediment faster

compared with the A

nucleocapsids.

In

addition,

B

nucleocapsids

contain

a

major protein

com-ponent

(protein 22a) not found in A

nucleocap-sids. We have

also observed two different classes

of

nucleocapsids which sediment at

different

rates and appear to

correspond to

the A and B

groups.

SDS-PAGE

analysis

of

preparations of

the

slow-sedimenting

class of

nucleocapsids

clearly revealed

that

they possessed

relatively

lower amounts of

p40

than did preparations

containing the

fast-sedimenting

class

(Fig. 1 and

Hsw4ris

Nluc§eocap.S4t-.;;f',,;

r

'rx,tst

-e,i:

,r.:s.-

; r,c: p _*

a:

--er:--S

ses!J

F4 t_^

FIG. 1. SDS-PAGE analysis ofpolypeptidesand

'"I-labeledp40

fromdisruptedHSV-2 MSnucleocapsids. (A)Scanning densitometer tracing of Coomassie brilliant blue-stainedgel. (B)Autoradiogram ofgel

contain-ing

125I-labeledp40.

(C)Scanningdensitometertracing ofautoradiogram. Molecular-weightstandards used

included

,8-glactosidase

(130,000),phosphorylase a(92,000),ovalbumin (43,000), DNase I(32,000),and

myoglo-bin(17,000).

J. VIROL.

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NUCLEOCAPSID p40 FROM HSV 37

RECIPROCAL ANTISERADILUTON(bilo)

FIG. 2. Immunoprecipitation ofHSV-1and HSV-2 '251I-abeled p40 by homologous and heterologous guineapigantisera. 1251-labeledp40 (10,000cpm)from

HSV-1 (A) orHSV-2 (0) wasincubatedwith serial twofolddilutions ofantisera elicited againsteither HSV-1 (A)orHSV-2(B) p40. The reactionmixtures andconditionswere asdescribed in thetext.

3). This finding, together with similarities in

molecularweight, suggeststhatp40is thesame

asprotein22a and thatourslower-sedimenting

nucleocapsid preparations probably representA

nucleocapsids. To establish thatour

immunolog-icalprocedureswere assaying aprotein having

thepropertiesofprotein22a andp40,a

homol-ogous HSV-2 competition immunoassay was

performed by usingunlabeledpurified p40 and

preparationsofdisruptedfast(B-enriched)-and

slow(A-enriched)-sedimenting nucleocapsidsas

competing antigens (Fig. 4). Precipitationof

la-beledantigenwas50% inhibitedbythepresence

of about 1.5 ng of purified p40, 10 ng of

fast-sedimenting nucleocapsids, or 170 ng of

slow-sedimenting nucleocapsids.Thisassayindicates

thatp40accounts for about 15%of theprotein

found in

fast-sedimnenting

nucleocapsids and

about 1% of the

protein in

slow-sediinenting

nucleocapsids.

Very

similar

results were

ob-tained

in

homologous HSV-1

assays (data not

shown). Preparations of disrupted

HSV-1

nu-cleocapsids

did not compete inthe homologous

HSV-2 immunoassay, further

suggesting the

ex-istence of type-specific

antibodies to

p40.

These

assays

also show that

our

total

nucleocapsid

preparation contained predominantly

fast-sedi-menting (B) nucleocapsids, because

the total

nucleocapsid

preparation contained about the

same amount

of

p40

as

did

the B

nucleocapsid-enriched preparation.

Homologous

competition

HSV-1

and

HSV-2 immunoassays for p40 in cell

ex-tracts.

In

the

homologous HSV-1 immunoassay

(Fig. 5A), unlabeled

extracts

of

HSV-l-infected

celLs inhibited

precipitation of

HSV-1

'WI-la-beled

p40, whereas

extracts

of cells infected with

HSV-2

or

SA8 virus

competed to

only

a

small

extent.

No

competition

was

observed with

ex-tracts

of

uninfected cells

or

PRV-infected

cells.

In the

homologous HSV-2 immunoassay (Fig.

5B),

extracts

of HSV-2-infected

cells inhibited

i

FIG. 3. SDS-PAGEanalysis ofthepolypeptides of theslow-sedimenting nucleocapsids. PurifiedHSV-2 MS slower-sedimenting nucleocapsids (30ug) were

subjectedtoelectrophoresis, andascanning densi-tometertracingwasmadeoftheresultingCoomassie brilliant blue-stained polyacrylamide gel. Two classesof nucleocapsidswerepurified byseparately

collecting the slow (A nucleocapsid-enriched)- and fast (B nucleocapsid-enriched)-sedimenting bands

formed afterthesucrosegradient centrifugation

pro-cedure described in thetext. Thenucleocapsidswere

diluted with PBS, sedimented by centrifugation at

23,000rpmfor 1 h at4°Cin a SpincoSW27rotor,

andsuspendedin 0.01 MTris-hydrochloride-0.001M EDTA, pH 7.5. After sonic treatment at 0°C, the nucleocapsids were layeredonto a 16-ml 45to 65% (vol/vol) Renografin 76gradientpreparedin0.01 M

Tris-hydrochloride-.001MEDTA, pH7.5, and

cen-trifugedtoequilibriumat25,000rpmfor3 hat4°Cin

aSpincoSW27.1 rotor(12). The slow- and fast-sedi-menting nucleocapsids formed lighter and heavier

bands, respectively, aftercentrifugationin the Reno-grafin 76gradient (2, 11). Theslow-sedimenting

nu-cleocapsidswerediluted with 0.01 M Tris-hydrochlo-ride-0.001 MEDTA, pH 7.5,pelleted by centrifuga-tionat23,000rpmforIh at4°CinanSW27.1rotor,

andfinally suspendedin water.

a

z

0

z0

C

F. z

a

5,

A Nucloocapsids

p156 p50 p32 p25 p12

VOL. 29, 1979

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[image:4.501.58.214.53.410.2]

38

HEILMAN ET AL.

0

20

0~~~~ ~ ~ ~~

0 2 3 4

[image:5.501.69.267.54.276.2]

Competing Nucleocapsid Protein(loglong) FIG. 4. Homologous competition immunoassay for p40in whichpurified p40anddisrupted nucleocap-sidswereusedascompetingantigens. Total

nucleo-capsids, purified asdescribedin thetext, and

slow-andfast-sedimenting nucleocapsids, purifiedas

de-scribed in thelegendtoFig. 3,weredisruptedin 0.01

M Tris-hydrochloride (pH 7.8)-0.01 M NaCI-0.1% Triton X-100containing1%SDS and 5 mM DTTby heating at100°C for5min. Thepreparationswere

then dilutedfourfoldwith the same buffer without

SDS orDTT and tested atserial twofolddilutions

for abilitytocompete with '25I-labeledHSV-2 MSp40 (10,000 cpm) for binding limiting concentrations of guinea pigantiserum(diluted1:5,000)elicitedagainst HSV-2 MSp40. Thecompeting antigenswereHSV-2

MSp40 (0; purified asdescribed in the text),

dis-ruptedtotalnucleocapsids ofHSV-1 MAL (Q) and

HSV-2 MS (0), and disrupted slow (V)- and fast-sedimenting(A) nucleocapsids ofHSV-2 MS.

precipitation, whereas uninfected cell extracts

and extracts of HSV-1-, PRV-, or SA8

virus-infected cells didnotdetectably compete. The

homologous assays showed that about 5 x

104

HSV-1-orHSV-2-infectedcellsextracted late in

infectioncontainedapproximately0.2jigofp40.

The reactivities ofp40in thesehomologous

as-saysindicatedthat theproteinsfromboth HSV

typespossesstype-specificimmunological

deter-minants. To verify that a single protein was

beingcharacterizedbythe aboveimmunoassays,

unlabeled nucleocapsid polypeptides were

sub-jectedtomolecularsizing analysis by gel

filtra-tion in thepresenceof 6 Mguanidine

hydrochlo-ride, and individualcolumn fractionsweretested

by competition immunoassay. Reactivityinthe

homologous HSV-1 p40 assay (Fig. 6A) and

HSV-2 p40assay (Fig. 6B)chromatographedas

a single homogenous peak at approximately

40,000molecular weight. The specificity of the

p40 antiserum reaction was also tested after

separating nucleocapsid polypeptides by

SDS-PAGE (Fig.

7),

because gel filtration did not

provide good separation of the p50, p40, and p32

components.After thepolyacrylamide gels were

stained and sliced into segments, the

polypep-tides wereeluted, and the eluants were tested by

competition

immunoassay. Reactivity

was

only

eluted from thegel segments containing the p40

protein.

To characterize the competing

antigen with

respect to

charge,

an HSV-2 p40 preparation

was

analyzed

bycolumn isoelectric focusing. As

shown inFig. 8, awell-defined peak of competing

reactivity was found at pH 6.7, which provides

further

support for the high specificity of our immunoassay.

Heterologous

competition immunoassay

for HSV-1 and HSV-2

p40.

The

precipitation

of

heterologous p40

by

guinea

pig antisera at

relatively

high

titers

(Fig.

2)

suggested that these

nucleocapsid proteins also contain shared

deter-minants. To

test

this

possibility, competition

immunoassays with HSV-1 p40 (Fig.

9)

and

HSV-2 p40 (data

not

shown)

were

performed

with

heterologous antisera.

In both cases, the

results

were

essentially the

same. In an assay in

which

antibody

to HSV-1

p40

was utilized at

limiting dilution for precipitation

of

1251-labeled

HSV-2 p40,

extractsof

cells

infected with either

HSV-1

or

HSV-2 competed

with comparable

high efficiencies (Fig.

9).

Similar

highefficiencies of

competition

were

observed

whenHSV-1 and

HSV-2 nucleocapsids

were used as competing

antigen (data

not

shown). Uninfected cell

ex-tracts

and

extracts

of

cells infected

with PRV

were not

reactive,

whereas extracts of

cells

in-fected

with

SA8

viruscompeted to a

final

extent

of 50%.

Immunoprecipitation of

p40 with

ab-sorbed

sera. To

further

establish that

p40

con-tains both

type-specific

and

cross-reactive

deter-minants, immunoprecipitation

tests were

per-formed

with sera

absorbed

with

unlabeled

HSV-1 or

HSV-2 disrupted nucleocapsids

(Fig. 10).

Absorption of anti-HSV-2

p40

with HSV-1

nu-cleocapsids

did not measurably reduce the

se-rum titer against

'25I-labeled

HSV-2

p40,

whereas

absorption

with

HSV-2 nucleocapsids

caused a substantial reduction in titer.Similarly,

a

slight decrease

intiter against HSV-1

p40

was

detected after

absorption

of HSV-1 antiserum

with HSV-2

nucleocapsids,

whereas a very large

reduction in titer was observed afterabsorption

with HSV-1

nucleocapsids

(data not shown).

Theseresults suggest that

p40

induces the

pro-duction of one

class

of antibodies which react

with type-specific

determinants

and a second

class which bindcross-reactive determinants.

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NUCLEOCAPSID p40 FROM HSV

I

2

0

0

i

-I

c31d

I-0

40

20

Fraction Number

FIG. 6. Specificity of competition

radioimmunoas-sayfor HSV-1 (A) and HSV-2(B)p40'sby guinea pig antisera. Nucleocapsids (1 mg) were disruptedand

appliedtoaBio-Gel A-5agarosecolumn(1.5 by90

cm) in thepresenceof6Mguanidine hydrochloride

and 0.01 M DTT. Fractions (1.0ml) were coUected,

dialyzed against 0.01 M Tris-hydrochloride (pH

7.8)-i mMEDTA-0.1% TritonX-100, andtested at

serialtwofolddilutionsfortheirabilitytocompete in thehomologous p40 immunoassays. Results are

ex-pressedasthepercentageoftotalantigenic reactivity

in eachcolumnfractionandarebasedonthe dilution atwhich 50% displacement of the

"*I-labeled

antigen

was achieved. Mokcular-weight markers include blue dextran (>100,000), bovine serum albumin

(68,X000), Rauschermurineleukemiavirusp30(30,X000),

avian leukemiavirusp12(12,000), andbromophenol blue(<1,000).

Reiprocal Antigen Dilution(logio)

FIG. 5. Homologous competitionimmunoassayfor

HSV-1 and HSV-2 p40. Unlabeled infectedcell

ex-tractsweretestedatserial twofold dilutionsfor their abilitytocompete with(A)

"LI-labeled

HSV-1 MAL p40(10,000 cpm) for binding limiting concentrations of guinea pig antiserum (diluted 1:400) directed against HSV-1p40, and (B)

125I-labeled

HSV-2 MS p40(10,000 cpm) for binding limitingconcentrations of guinea pig antiserum (diluted 1:5,000) directed against HSV-2p40. CeUswere infectedata

multi-plicityof infection of Iasdescribedin thetextand

harvestedatthe timewhenmaximumcytopathic ef-fects wereobserved. This wasat18h postinfection with theHSV-1strainsandat36h postinfectionwith HSV-2strains, PRV, and SA8 virus. Cell extracts

werepreparedbysuspending approximately2.5x 106

cells in 0.5mlof PBS,subjectingthemtosonic treat-ment,andaddingSDS andD1T tofinal

concentra-tionsof1% and 5

mM,

respectively.The extractswere

heatedat IOOOCfor2min, debriswas removed by centrifugation (1,2(M) xgfor15min),andthe extracts were diluted in 0.01 M This-hydrochloride (pH 7.8)-0.01 MNaCl-0.1% TritonX-100. Undiluted

com-petingantigenin the reactionmixturerepresentsan extractofabout 5xIO ceUs.Thecompetingantigens

wereextractsofcellsinfectedwith HSV-1 MAL(0), HSV-1 14-012 (0), HSV-2 MS (A), HSV-2 Savage (V), HSV-2333 (A), SA8virus (L), PRV(U), and

uninfectedVero cells(0).

C ->

I I

Is

F

I I I I

D->

30 50 70 90 10

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1979

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40

HEILMAN ET AL.

FractionNumber

FIG. 7. Specificity of competition radioimmunoas-say for HSV-2 p40 as shown by SDS-PAGE. Dis-ruptedHSV-2 MSnucleocapsids (approximately30

ug) were subjected to electrophoresis in cylindrical

12%polyacrylamide gels (7), stained with Coomassie brilliant blue, and scanned for absorbance (A). The gelwaswashed severaltimes with water, frozen, and sliced into 1.35-mm segments. Protein was eluted overnightat roomtemperaturefrom each segment in 0.5mlof elutionbuffer containing0.1MTris, pH7.8,

0.4MNaCl,and 0.1% SDS. Eachfractionwastested

forcompetition(B)inahomologousassayfor HSV-2

MSp40in serialtwofolddilutionsasdescribed in the

text.Resultsareexpressedasthe percentageof total antigenic reactivity in eachfraction andare based onthedilutionatwhich50%o displacementof the

1"5I

labeledantigenwasachieved.

Analysis

of human

sera

for antibodies

to

HSV-1 and HSV-2

p40's.

In

view of the

sen-sitivity of the

immunoprecipitation procedure

and its

ability

to

discriminate 1 and

HSV-2

p40's,

it

was

of interest

toassess

the

potential

application

of this

test to

analysis

of

human

sera

for antibodies directed

against

either

HSV-1

or

HSV-2.

For this purpose, aseries ofsera

from

individuals with

suspected

HSV-1

or

HSV-2

ex-posure were tested

for

immunoprecipitation

of

'25I-labeled

HSV-1 and HSV-2

p40 proteins,

both

directly

and

after

absorption

with

unlabeled

de-tergent-disrupted

nucleocapsids.

We had

anticipated

from the

p40

immunoas-say results with

guinea pig

antisera that we

would be able todivide the humanserum

sam-ples

into four

categories: (i) HSV-negative

sera,

(ii) anti-HSV-1

sera,

(iii)

anti-HSV-2

sera, and

(iv) anti-HSV-1

and -HSV-2 sera.

Our

prelimi-nary

findings

(Table 1)

indicated that each of

the sera tested

clearly

fall

into one of the four

expected

categories.

Assignments

ofthese sera

tothe category of

anti-HSV-1

positive

or

anti-HSV-2

positive

wasbasedonthe

demonstration

that

absorption

of the serum with one antigen

caused a substantial decrease in its ability to

precipitate

1251-labeled

p40,

whereas absorption

with the other

antigen resulted in

only

a small

reduction in its

precipitating

activity. The

exist-ence of

human antibodies

to

the cross-reactive

determinant(s) of p40

was

suggested

by

the

find-ings that both

the

anti-HSV-1 and

anti-HSV-2

sera

precipitated the labeled

heterologous

pro-tein and that these

precipitations

were

blocked

by

absorption with either

homologous

or

heter-ologous nucleocapsids.

DISCUSSION

The

approximately

40,000-molecular-weight

(p40)

polypeptides

from HSV-1 and

HSV-2

nu-cleocapsids

were

purified, and antisera

were

pre-pared for utilization

in

direct and

competition

radioimmunoassays. The

findings

indicated that

the

p40's of HSV-1 and HSV-2

possess

both

cross-reactive and

type-specific antigenic

deter-minants. The various

procedures

carried

out to

verify the

purity of the p40 isolates

suggests

that

both the

cross-reactive

and

type-specific

deter-minants

reside

on the same

polypeptide

mole-cule.

This is

similar

to

what

has been

described

for

polypeptides of mammalian oncornaviruses

(10).

50

I-4

z

m

4c

40

30

20

10

o0

6

2

10 20 30

Fraction Number

FIG. 8. Isoelectricfocusing analysis of HSV-2p40. Purified HSV-2 MS nucleocapsids (5 mg)were

dis-rupted and appliedtoan agarose-guanidine

hydro-chloridecolumnasdescribed in the legendtoFig.6.

After dialysis against 0.01 MTris-hydrochloride(pH

7.8)-i mM EDTA-0.01% Triton X-100, fractionswere

analyzed bySDS-PAGEasdescribed in thetext.The

p40-containing fractions werepooled and analyzed

by isoelectric focusing. Focusing wasperformedat

300 Vfor 48 h in the pHrangeof3.5to10 ina

110-ml-capacity column (LKB Instruments, Inc.,

Rock-ville, Md.) (10). Fractions (3 ml) werecollected,

di-alyzed, and analyzed by competition immunoassay asdescribed in thelegendtoFig.6.

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NUCLEOCAPSID p40 FROM HSV 41

i

20 la

o

02

260

IC

04

-IO

20~~~~

4 3 2 1

ReciprocalAntigenDilution(log91)

FIG. 9. Heterologous competition immunoassay for HSV-2 p40. Cellextracts,preparedasdescribed

in thelegendtoFig. 5, weretestedatserial twofold dilutionsfor abilityto competewith'25I-labeled HSV-2MS p40(10,000 cpm)for binding limitingamounts

ofguinea pig antiserum elicited against HSV-1 MAL p40.The antiserumwasusedatadilution of 1:1,000,

which immunoprecipitated about 30% of the 'MI-la-beled HSV-2 MS p40 antigen (Fig. 2). The competing antigens, which were those used in the experiment

describedinthelegendtoFig.5,wereextractsofcells infected with HSV-1 MAL (0), HSV-1 14-012 (0), HSV-2 MS (A), HSV-2 Savage (V), HSV-2333 (A), SA8 virus(L), PRV (U), anduninfected Vero cells (01).

In agreement with studies

reported by others

(Cohen

et

al.,

Abstr. Annu. Meet. Am. Soc.

Microbiol.

1978,

S42,

p.

219),

the

p40's

of

HSV-1 and HSV-2 were localized to the nucleus of

productively

infected cells

by

immunofluores-cent

staining (unpublished

data).

The

competi-tion

immunoassays

didnotdetect

intratypic

dif-ferences in the

antigenic specificities

of

p40's

from the

different strains of HSV-1 or HSV-2

studied.

Antigens produced

in PRV-infected

celLsfailedtoexhibitdetectable

cross-reactivity

with

the

p40's

of HSV-1 orHSV-2. In contrast,

cross-reactivity

between HSV-1 and HSV-2

p40's

and

antigens produced

incells

productively

infected

with

SA8

viruswas

observed,

which is

consistent with

previous

reports

(6, 16)

that

these

viruses share

cross-reacting

structural

an-tigens.

The

findings by competition

radioim-munoassay suggest that

SA8

virus and HSV

o AAA

REIOAL A 0

10. a

ew

z

40 at

2040

1 2 3 4

[image:8.501.270.429.53.284.2]

RECIPROCAL ANTISERADILUTION(Ioglo)

FIG. 10.

Immunoprecipitation

awsay

for

`~I-la-beledHSV-2

p40 by

unabsorbed and

nucleocapsid-absorbedantisera.

Limniting

amounts

of "MI-abeled

HSV-2

p40 (10,0(10

cpm)

were reacted with

twofold

dilutionsof guineapig anti-HSV-2p40 serum that was either unabsorbed (0) orabsorbed witheither HSV-1 (A) orHSV-2(0) nucleocapsids.Adsorption

wasaccomplished by incubatingundiluted antiserum

withdisrupted nucleocapsids

(20

pg) for2h at37°C.

The nucleocapsids were disrupted by heating at

100°C for5min in the presenceof 0.1% SDSand 1

mM DTT.

share cross-reactive

antigenic

determinants.

The

development

ofsensitive

radioimmunoas-say

procedures

for

specific HSV proteins

should

allow for

more

extensive

immunological

studies

with this

virus

group.

Utilizing

the

p40

assays

as

described here, for

example,

will

allow the

rapid

typing

of

new

HSV isolates.

Furthermore,

as

observed in

our

preliminary

studies with human

sera, the

use

of the

p40 immunoassay

should

allow for the

accurate

determination of

antibody

levels in human

sera to

HSV-1 and HSV-2

type-specific

and

cross-reactive

antigens.

Such studies

will facilitate the determination of

antibody

lev-els in human

sera

and

fluctuations which may

occur

in

these

antibody

levels

during

various

disease

states

in

which HSV has been

impli-cated. Studies

are

presently underway

to

expand

the

available

immunoassay

procedures

toother

HSV-1- and HSV-2-induced

proteins,

which

should further enhance the

number

of

variables

amenable

to

study.

ACKNOWLEDGMENT9

Thisworkwassupported byPublic Health Servicecontract N01-CO-75380 from the Virus CancerProgram,National Can-cerInstitute.

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42 HEILMAN ET AL.

TABLE 1. Precipitation of '25I-labeledHSVp4O by human seraa

%Precipitation of p40 from:

Human'serum HSV-1 MAL HSV-2MS

Humanb serum____________ _____

Absorbed Absorbed Absorbed Absorbed

Unabsorbed HSV-1 HSV-2 nabsorbed HSV-1 HSV-2

HSV negative

101 3.7 3.8 5.1 1.8 2.0 2.3

102 3.2 3.3 3.9 2.1 2.0 1.7

HSV-1positive

1689 74.8 19.3 69.7 49.9 4.2 3.9

1690. 77.2 17.6 66.0 44.6 5.8 5.5

1697 73.3 16.7 70.6 65.3 3.0 3.3

HSV-2positive

1691 36.4 11.5 11.7 77.1 59.1 5.1

1694 66.2 18.7 13.2 87.0 75.5 7.2

1719 39.4 12.0 12.8 92.1 76.4 5.5

HSV-1 and HSV-2positive

1696 61.0 19.0 40.2 70.6 51.8 5.4

1698 69.9 19.8 34.6 83.0 76.5 6.1

1705 38.5 11.1 27.1 40.4 28.8 3.3

a

'25I-labeled

p40

(10,000 cpm) was incubated with sera asdescribed in the text.Goat anti-human

inmmuno-globulin Gwasusedasthe second antibody. SerawereabsorbedbyincubationwithdisruptedHSV-1or HSV-2nucleocapsids (20fg)for1hat37°Cbefore the addition of'251I-labeledp40. The boldfacing indicatessignificant

precipitation of '251-labeled p40 after absorption with the putative heterologous antigen and serves as an

indicator oftype-specific reactivity.

bThe human sera wereassayedat a1:10dilution. The HSV-negative serawerecharacterized assuchby

neutralization and immunofluorescence. The anti-HSV-positive serawere obtained from K. Hsu, Columbia

University.

Wethank E. Ambush and M.Chakrabartyfortheir excel-lenttechnicalassistance.

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