Shared antigenic determinants between two distinct classes of proteins in cells infected with herpes simplex virus.

JOURNAL OFVIROLOGY, Sept.1980,p.644-652 Vol. 35,No. 3 0022-538X/80/09-0644/09$02.00/0

Shared

Antigenic

Determinants Between Two Distinct Classes

of Proteins in Cells

Infected

with Herpes

Simplex

Virus

MARTIN

ZWEIG,`*

CONRAD J. HEILMAN,JR.,' HARVEYRABIN,2ANDBERGE HAMPAR3

Carcinogenesis IntramuralProgram,' BiologicalCarcinogenesis Program,2 and Laboratory of Molecular Virology,NationalCancer Institute,3 Frederick Cancer ResearchCenter, Frederick, Maryland21701

Guineapig antisera and mousemonoclonal antibodiesagainst a

40,000-molec-ular-weight nucleocapsid protein (p40)

of

herpes simplex

virus types 1 and 2

immunoprecipitated

40,000- and

80,000-molecular-weight

classes ofsoluble pro-teins from infected cell extracts. The soluble

40,000-molecular-weight

protein

class

(intracellular p40) appeared

asacluster of threetofourclosely spacedbands

ofproteins having molecular weights ranging between 39,000 and 45,000,whereas

thesoluble

80,000-molecular-weight

protein class(intracellular p80) appearedas

adoublet of bands. The peptide map of intracellular p40 closely resembled the

maps of the p40 andp45 proteins of nucleocapsids, but it showed bothdifferences

and similarities when compared with thepeptide mapofintracellularp80.

Pulse-chase

experiments

suggested that intracellular p80 was not a precursor of

intra-cellular p40. We conclude that

the intracellular p40 and p80

protein

classesshare common antigenic determinants, presumably reflecting similar amino acid se-quences, although they have distinct differences inproteinstructure.

In our

laboratory

we have been

engaged

in

studying

the

nucleocapsid proteins

of

herpes

simplex

virus type 1

(HSV-1)

andtype 2

(HSV-2).

We have shown by competition immunoas-says that a

40,000-molecular-weight

polypeptide, p40, which is a

major

component of these

nu-cleocapsids,

possesses

both

type-specific and

cross-reactive

antigenic

determinants

(7).

Ra-dioimmunoprecipitation

studies with

monospe-cific antisera and

monoclonal antibodies have

indicated

that

p40

shares at least some of these

determinants

withanother

nucleocapsid

protein,

protein

p45

(14).

The

finding that nucleocapsid proteins p40

and

p45

share

antigenic determinants led

us to

examine infected

cells for the

presence

of

other

proteins

which

might

have the same

determi-nants.

Such

proteins

might be

precursors,

poly-proteins,

or

other

types

of derivatives which

could indicate

important relationships

among these

proteins.

Wefound that

antibodies against

nucleocap-sid

p40 immunoprecipitated

40,000- and

80,000-molecular-weight classes of

proteins

from in-fected cell extracts.

The

40,000-molecular-weight class

(intracellular p40) appeared

to in-clude the

nucleocapsid

proteins p40

and

p45, whereas the

80,000-molecular-weight

class

(in-tracellular

p80)

was notfound in

nucleocapsid

preparations. Pulse-chase

experiments

failed to show a

precursor-product relationship

between

intracellular

p80

andintracellular

p40.

The pep-tidemaps of these two classes of

proteins

showed both

differences

andsimilarities.

MATERIALS AND METHODS

Cells, viruses,andantibody preparation. HSV-1 strain MAL andHSV-2 strain MS were grown in Vero cells as previously described (7). Guinea pig antisera against HSV-1 and HSV-2 p40's were

pre-pared byimmunizing animals with purifiedp40protein obtained frompolyacrylamide gels (7). Ascites fluids containing monoclonal antibodies against p40 were produced by intraperitoneal inoculations of hybridcell

lines 1D4 (whichsecretesanti-HSV-1 antibodies) and 3E1 (whichsecretesanti-HSV-2 antibodies) (14) into

BALB/cmiceprimed with Pristane (Aldrich Chemical Co., Milwaukee, Wis.),asdescribedby Lostrum et al.

(9).

Purificationof virions. Virions were purified

es-sentially as described by Spear and Roizman (13).

Cells infected withHSV-1 orHSV-2were harvested 24hpostinfection, suspended in2volumes of1 mM dibasic sodiumphosphate (pH8.2)containing0.1 mM

phenylmethylsulfonyl fluoride, and allowed to swell

for10minat0°C.Thecellsweredisrupted by dounce homogenization,andthe nuclei werepelletedby cen-trifugationat800xgfor10min andstoredat-70°C until used for the purification of nucleocapsids (see below). Debris was removed from the cytoplasmic fraction bycentrifugation in aSorvall SS34rotor at 8,000 rpm for5min. The virions in thecytoplasmwere sedimentedby centrifugation at 60,000 x gfor 1 h, suspendedin 1 mM dibasic sodium phosphate, and subjectedtosonic treatment. After incubationinthe presenceofDNase (50,ug/ml)andRNase (50

tLg/ml)

for30min at room temperature, the virionsamples werelayeredonto34-ml5to 30%(wt/vol) dextranT1O

(Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) gradients in 1 mM dibasic sodium phosphate and centrifugedfor 1 h at20,000 rpmin aBeckmanSW27 rotor. The virion-containing band wascollected, di-644

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lutedapproximatelyfourfold with 10 mM Tris-hydro-chloride (pH 7.6)-i mM EDTA (TE buffer), and pel-leted by centrifugation at 25,000 rpm for 1 h in a Beckman SW27 rotor. The virionpellet was suspended in asmall volume of TE buffer and was layered onto an11-ml10to50%(wt/wt)potassiumtartrategradient in TEbuffer. Centrifugation of the gradient was per-formed with aBeckman SW41 rotor for 2 h at 25,000 rpm. The virusband was collected, dialyzed against TEbuffer, and storedat-70°C.

Purification of nucleocapsids. Nuclei of infected cellsobtained asdescribed above were suspended in about 2volumes of 0.1 M Tris-hydrochloride (pH

8.0)-1.5mMMgCl2-0.1mMphenylmethylsulfonyl fluoride. The nuclei were lysed by adding sodium deoxycholate to a final concentration of0.5%, followed by sonic treatment.The nuclear lysates were incubated at room temperature for30 min inthe presence of50,igof DNase perml;thiswasfollowed by centrifugation at 8,000 rpmfor5mininaSorvall SS34 rotor to remove debris.Nucleocapsidswerepurifiedfromclarified nu-clear lysatesbycentrifugation through 35% (wt/vol) sucrose,followedby centrifugationina10to40% (wt/ wt) sucrosegradient, as describedpreviously (6, 7, 13).

Radiolabeling ofcells. Cells were washed once with methionine-freeEagleminimalessential medium containing 5%dialyzedheat-inactivatedfetal calf se-rumand thenlabeled with 100

,uCi

of[3S]methionine

(800 to 1,200 Ci/mmol; Amersham Corp., Arlington

Heights,Ill.)perml in thesamemethionine-free me-dium for 1 to 4h. The cell sheetwas thenwashed twice withice-cold Tris-buffered saline (pH 7.4) and

scraped, and the cells weresuspended in cold Tris-buffered saline. The cells were sedimented by

centrif-ugationat800xgfor 10 min, and the cellpelletswere storedat-70°Cuntilused.

Preparation ofcellextractsanddisrupted vi-rus particles for immunoprecipitation. Cell ex-tractswereprepared by suspending the

[3S]methio-nine-labeledcells in buffer A (0.1 M Tris-hydrochlo-ride

[pH

8.0], 10%[vol/vol]glycerol, 0.5% Nonidet P-40, 0.5%sodium deoxycholate,0.2mM phenylmeth-ylsulfonyl

fluoride)

andincubating them for1h at4°C

withshaking.The extracts were clarified by

centrifu-gation at60,000xgfor1 h.[3S]methionine-labeled

virionsandnucleocapsidswere disrupted by heating at 100°C for 5min in 0.5% sodium dodecyl sulfate

(SDS)-2.5%

/i-mercaptoethanol-0.05

M Tris-hydro-chloride (pH8.0),followedby a10-fold dilution with buffer A (14).

Immunoprecipitation and SDS-polyacryl-amide gel electrophoresis. Antibody (20

jl)

was incubated witha0.5-mlportion of eithercellextract ordisrupted virusparticles for 3 h at 4°Cand then further incubated for1hwith 0.12 ml ofa33%(vol/

vol) suspensionofprotein A-Sepharose CL-4B beads

(PharmaciaFine Chemicals, Inc.). Afterincubation, the beads werewashed with 0.5 MLiCl-0.1 M Tris-hydrochloride (pH 8.0)-1% B-mercaptoethanol, sus-pendedin anequal volume of 2% SDS-20% glycerol-5%

,8-mercaptoethanol-0.125

M Tris-hydrochloride (pH 6.8)-0.004% bromophenol blue, and heated at 100°C for 5 min, as previously described (14). The proteinswerethenseparated by electrophoresis on a 5to20%polyacrylamide gel gradient containing SDS

(7), and autoradiographs or fluorographs were pre-paredonKodakSB-5 X-rayfilm(1).Theabsorbances at 595 nmof the bands in theautoradiographs and

fluorographs weremeasured with a scanning densi-tometer (Transidyne General Corp., Ann Arbor, Mich.)

Peptide mapping. Proteins obtained from poly-acrylamide gelswerepartiallydigested with Staphy-lococcusaureusV8protease(Miles Laboratories, Inc.,

Elkhart, Ind.), and the peptides were analyzed by

SDS-polyacrylamide gelelectrophoresisessentiallyas described by Cleveland et al. (4) and Bordier and

Crettol-Jarvinen (2).Afterseparation by

electropho-resis,[3S]methionine-labeled proteinswerelocated in driedunfixedgels by alignmentwithautoradiographs,

and rectangular segments (0.5 by 1.5 cm) of lanes containing theseproteins were excised from the gel

slabs and incubated in 0.125 M Tris-hydrochloride (pH 6.8)-0.1%SDS-1 mM EDTA for 30 minatroom temperature.Thegelsectionsweredrained andplaced

in sample wells (width, 2 cm) of second-dimension

polyacrylamide gels,whichwerecomposedofa7-cm 5 to20%gradient gelbeneatha3-cm4%stackinggel.

The gelsegmentswere orientedsothat their bands wereparalleltothedirection ofelectrophoresisin the

second-dimension gel, and then they were covered withmelted(55°C)1% agarose in 0.125 M

Tris-hydro-chloride (pH 6.8)-0.1% SDS-1mMEDTA. S.aureus V8 protease in 0.125 MTris-hydrochloride (pH

6.8)-0.1% SDS-1 mM EDTA-10%glycerol-0.004%

bromo-phenolbluewasoverlaidonthehardened agarosegel,

andelectrophoresiswasconductedatlowvoltage(25 V) for 20 h to allow partial proteolytic digestion to

occur during the stacking phase of

electrophoresis.

Afterelectrophoresis,thegelswereprocessedfor

fluo-rographyonKodakSB-5 X-rayfilmbythe method of Bonner andLasky (1).

Competition

immunoassays.Purified virions and

nucleocapsids were disrupted in 0.01 M

Tris-hydro-chloride (pH 7.8)-0.01 M NaCl-0.1% Triton X-100 containing 1% SDS and5mMdithiothreitolbyheating

at 100°Cfor5min.The preparationsweretestedat

serial twofold dilutions in the same buffer without

SDSordithiothreitolforabilitytocompetewith

'25I-labeled HSV-1 strain MALnucleocapsid p40 (10,000

cpm) forbindinglimitingconcentrations of

guinea

pig

antiserumagainstHSV-1

nucleocapsid

p40.Antiserum was used at a dilution of 1:400, which precipitated approximately 35% of the

l"I-labeled

p40.

Prepara-tionsof

'"I-labeled

p40,theantiserum,and the

reac-tion condireac-tions have been described

previously (7).

RESULTS

Reaction of

guinea pig

antiserum

with

cell

extracts. HSV-2-infected and

uninfected

cells

labeled

with

[3S]methionine

were

dis-rupted

with

detergents

and were clarified

by

high-speed

centrifugation,

which

sedimented

vi-rus

particles and insoluble proteins.

A

large

number

of

soluble

virus-specified

proteins

were

observed

in

infected

cell extracts

by

SDS-poly-acrylamide

gel

electrophoresis

(Fig.

1,

lane

A),

asevidenced

by

their absence in

uninfected

cell

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646 ZWEIG ET AL.

A

B

C

D

E

F

G

J. VIROL.

-

p155

:_

_

_

p50

*. ....:..

._SUhi - p40

0e-

p32

_

p12

FIG. 1. SDS-polyacrylamide gel electrophoresis of soluble cell extractproteins immunoprecipitated by guinea pigantiserumagainstHSV-2nucleocapsid p40. HSV-2- andmock-infected cells werelabeled with [35S]methioninebetween 18 and 22 hpostinfection. Extractswerepreparedandreactedwith eitherguinea

pig antiserum against HSV-2 nucleocapsid p40orcontrolguinea pig serum. Proteinsfrom cellextracts,

immunoprecipitates, and nucleocapsids were separated by electrophoresis, and an autoradiograph was prepared.LaneA, proteins ofHSV-2-infectedcellextracts;laneB,theproteins of mock-infectedcellextracts; lanes C andD,proteinsprecipitated from HSV-2-infectedcell extractsbyguineapigantiserumagainst HSV-2nucleocapsid p40 (lane C)andby control nonimmuneguinea pigserum(lane D); lanes E andF, proteins precipitated from mock-infectedcellextractsby guinea pigantiserumagainstHSV-2nucleocapsid p40 (lane E)andbycontrol nonimmuneguinea pigserum(lane F);laneG,proteins ofpurified

[3"SJmethionine-labeled

HSV-2 nucleocapsids. Themajornucleocapsidproteinsaredesignatedatthe right ofthe autoradiograph. Thep45 band is the minor bandimmediately beneath p50.

extracts (lane B). Guinea pig antiserum against the HSV-2 nucleocapsid p40 protein immuno-precipitated two classes of viral proteins from infected cellextracts (laneC); thesewere

intra-cellular p80, which appearedas aband doublet,

and intracellular p40, which appeared asthree orfour closelyspaced proteins encompassinga

molecular weight range between 39,000 and

45,000. This antiserum didnotprecipitate these protein classes from uninfected cell extracts

(lane E), and nonimmune guinea pigserum did

notprecipitate them from eitherinfected (lane D) oruninfected (lane F) cellextracts. Compa-rableresultswereobtained with HSV-1-infected

cellextractsand antiserum against HSV-1 p40

(see below). Labeled proteins were not immu-noprecipitated from infected cells incubated

with['4C]glucosamine, suggesting that intracel-lularp40 and p80werenotglycoproteins (data

notshown).

Synthesis of intracellular p40 and p80 afterinfection. Inanexperiment in whichcells were pulse-labeled with [35S]methionine at in-tervalsafterHSV-2infection, synthesis of intra-cellular p40 and p80 was first detected by

im-munoprecipitation with guinea pigantiserum at

about 3 h postinfection (Fig. 2). The rate of

production of these protein classes remained roughlyconstantbetween6 and 24 h postinfec-tion, but considerablylessintracellularp80was

usually precipitated at 24 h postinfection.

Al-though thecauseof thisreductioninprecipitable intracellular p80 is uncertain, substantial cyto-pathic effects wereevidentat this latestage in

infection, and intracellularp80 may have been

selectivelylostinto themediumordegraded by

proteases.Antiserumwasreactedwith extracts

of infectedcells whichwerepulse-labeledfor15

min and then chased forupto4h with isotope-free medium (Fig. 3).

SDS-polyacrylamide

gel electrophoresis showed that theratio of the

in-tensities of the bands of intracellular p40 and

p80 remainedconstantduringthe chaseperiod, suggestingthat intracellularp40andp80didnot

serve asprecursorstooneanother.

Reaction of guinea pig antiserum with purified proteins. To verify that intracellular p40 and p80 possess common antigenic

deter-minants, each protein class was purified from

polyacrylamide gels and was successfully

im-munoprecipitated by guinea pig antiserum

against nucleocapsid p40 (Fig. 4). Therefore, it

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[image:3.510.88.403.72.295.2]

SHARED ANTIGENIC DETERMINANTS OF HSV PROTEINS 647

Hours Post-infection

1

2

3

4

5

6

7

8

-

p80

-

p40

9

10

11

12

16

24

- _I

-

p80

[image:4.510.57.249.79.323.2]

. ~~ ~ ...

FIG. 2. Synthesisofintracellular p40 andp80after

HSV-2infection.Cellswerelabeled with

[35S]methi-oninefor30min,terminatingattheindicated times

after

infection.

Cell extracts were

prepared

and

re-acted withguinea pigantiserumagainstHSV-2

nu-cleocapsid p40. Theproteinsinthe

immunoprecipi-tates wereseparatedby

SDS-polyacrylamide

gel

elec-trophoresis,andanautoradiographwasmade.

isunlikely that the precipitation of intracellular p80 from infectedcellextractswasduetospecific

ornonspecific bindingtointracellular p40. Fur-thernore, although the p40 protein exists in disulfide-linkedcomplexes in nucleocapsids (13), soluble disulfide-linkedcomplexes containing in-tracellularp40orp80werenotdetectedby

non-reducingSDS-polyacrylamide gel electrophore-sis(datanotshown).

Reaction of

monoclonal

antibodies with

infected

cell extracts.

Recently,

we

estab-lishedmousehybridcelllines whichsynthesize

monoclonalantibodiesagainst the p40 and p45 proteins of HSV-1 and HSV-2 nucleocapsids (14). Ascites fluids containing high titers of

mon-oclonal antibodieswere prepared andwere

re-acted withextractsofcells infected with either HSV-1 or HSV-2. The anti-HSV-1 p40

mono-clonal antibody produced by cell line 1D4

pre-cipitated intracellular p40 and p80 from only HSV-1-infected cell extracts (Fig. 5A). In

con-trast, the anti-HSV-2 p40monoclonal antibody produced by cell line 3E1 precipitated both

HSV-1 and HSV-2 intracellular p40 and p80

(Fig. 5B), although the homologous proteins

were

precipitated

at

higher

dilutions of

3E1

an-tibody

than

were

the

heterologous proteins.

Guinea

pig

antisera

against

the

nucleocapsid p40

proteins of

HSV-1 and HSV-2 also

precipitated

homologous

proteins

at

higher

antibody

dilu-tions, but the

differences in the dilutions which

precipitated the

homologous

and

heterologous

proteins

were not asgreat as

the

difference

ob-served with the

3E1

monoclonal

antibody (Fig.

5A and

B).

Peptide

maps

of

immunoprecipitated

proteins. The

finding

that

monospecific

anti-sera

and

monoclonal antibodies reacted with

HSV

polypeptides

having

different

molecular

weights

indicated

that these

n~~~

polypeptides

pos-a) U) u) cu

u) Xu cu s~

Cu . z ._

U U

C C 0

.T

E

Eg

E

o o

vr

o

7C)

CD N'4

p80-p40-

_p4O

...

~ ~

_ :..

FIG. 3. Autoradiogram of a polyacrylamide

gel

showingtheimmunoprecipitation

of

intracellularp40

andp80 fromcellspulse-labeledwith

[35S]methionine

and chased in nonradioactive medium. At16h

after

HSV-2infection, cellswereincubatedfor15min in

methionine-free medium. The medium wasthen

re-moved, and the cellswere

pulse-labeled

for

15min

with

[35S]methionine

in

fresh

methionine-free

me-dium. After the cellswere washed three times and incubated (chased) in complete nonradioactive me-diumfortheindicatedperiodsof time, cellextracts werepreparedand incubated with

guinea pig

anti-serumagainst HSV-2

nucleocapsid

p40.

The

immu-noprecipitatedproteinswereseparated by

SDS-poly-acrylamide gelelectrophoresis,and

autoradiographs

wereprepared. The relative intensities

of

thebands weremeasuredonscanningdensitometer

tracings

of

autoradiographs

preparedaftervarying intervals

of

exposure. VOL. 35,

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[image:4.510.289.433.236.488.2]

648

ZWEIG ET AL.

A B C

p80

_i

p40I

FIG. 4. Immunoprecipitation of purified intracel-lularp40andp80byguineapigantiserum against HSV-2 nucleocapsid p40. An extract of HSV-2-in-fectedcellslabeled with[35S]methionine between18 and22 hpostinfectionwasreactedwithguineapig antiserum againstHSV-2nucleocapsid p40,and the immunoprecipitatedproteinswereseparated by

SDS-polyacrylamide gel electrophoresis.Intracellularp40 andp80werelocated in thegelby alignmentwithan

autoradiograph, andgelsegmentscontaining intra-cellularp40andp80werecut out. Theproteinswere

eluted into bufferA and reacted witheitherguinea pigantiserumagainst p40orcontrolguineapig se-rum. The proteins in the immunoprecipitates were

separated bySDS-polyacrylamide gel electrophore-sis, andafluorographwasprepared. Proteinswere

precipitated by reacting purified intracellular p80 with antiserum against p40 (lane A) and control

serum(lane B)andbyreacting purifiedintracellular

p40withantiserumagainst p40 (lane C)and control

serum(lane D).

sess commonamino acidsequences. We tested

this hypothesis by an analysis of the peptide

mapsof

immunoprecipitated

proteinsafter

sep-aration by

SDS-polyacrylamide

gel

electropho-resis, partial digestion with S. aureus V8 pro-tease,and resolutionof thepeptides by

electro-phoresis inasecond

SDS-polyacrylamide

gel (2,

4). The peptide maps of the intracellular p40

andp80 classesrepresent thesumsoftheprotein bandscomprisingeachclass,since thevery

sim-ilarmobilities of thebandspreventedtheir clear

resolution andseparationinthefirst

electropho-resis

step.

The

peptide map

of

intracellular

p40

obtained

after

digestion

with 1

,ug

ofV8

protease

contained seven or

eight peptides,

which had

molecular

weights

ranging

from

40,000

(intact

intracellular p40)

to

8,000 (Fig. 6).

Nucleocapsid

p40

and

p45

had the same

peptide maps, except

that

they

lacked

the

8,000-

and

11,000-molecu-lar-weight

peptides.

The

nucleocapsid p45

digest

wasalso

deficient

in a

14,000-molecular-weight

peptide

whichwas

present

in the

digests

of

nu-cleocapsid

and

intracellular p40's. The digest

of

intracellular

p80 contained

intact

intracellular

p80

and

only

three smaller

peptides,

which

co-migrated

with

peptides found

in the

intracellular

p40

digest.

It is uncertain whether this

comigra-tion is a

reflection

of

the

presence

of identical

peptides

in

these

digests.

On the other

hand,

the

digests

of

intracellular and

nucleocapsid p40's

included peptides which did

not

comigrate

with

any

ofthe

intracellular p80 peptides. The

pep-tides

continued to show these common and dis-tinct

migration properties

when the

protein

classes were

digested

with

different

amounts of S. aureus V8

protease (Fig.

7).

Reaction

of

antibodies with virion

pro-teins.

Virions

of

HSV-1 and HSV-2

were

puri-fied

from

cytoplasmic extracts by

centrifugation

in

dextran T10

and

potassium tartrate

gradients.

Electron

microscope observations indicated that

the

HSV-1

virion

preparations contained

about 10 to 15%

unenveloped

nucleocapsids, whereas

the

HSV-2

preparations contained

as many as 50%

unenveloped particles. These

findings

are

reasonably consistent with

those obtained

by

Cassai

et al.

(3). Because of their

higher purity,

we

concentrated

our

efforts

on

analyzing

prepa-rations of

HSV-1 virions

to

determine whether

they

possess

polypeptides

immunologically

re-lated

to

p40. We

were not

able

to

identify

di-rectly

nucleocapsid p40 and

p45 in

virions

with

certainty

by electrophoretic

analysis because

of the

presence

of

proteins

having

a

migration

sim-ilar

to

that of

nucleocapsid

p40

(Fig.

8). Guinea

pig antiserum and

mouse

monoclonal antibody

against HSV-1

nucleocapsid

p40

precipitated

much smaller amounts of

nucleocapsid

p40and p45 from virion preparations than

from

prepa-rations

of

nucleocapsids, whereas

aprotein

hav-ing the

mobility of intracellular p80

was not

precipitated from

either virion or

nucleocapsid

preparations. We were not

able

tocompare

ac-curately

the amounts of p40 and p45 present

in

virions

with the

amounts

found

in

nucleocapsids

becausethe

preparations

may have had

differing

amounts

of

contaminating

extraneous

proteins.

DISCUSSION

Through

the use of monoclonal antibodiesand

J. VIROL.

on November 10, 2019 by guest

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650 ZWEIG ET AL.

A

INTRACELLULAR

p80 p40

I

0K :

80K-

I

28K -_

17K-

W_

1 1

K-p80 p40

[image:7.510.61.260.62.553.2]

INTRACELLULAR

FIG. 6. Peptide mapsof

guinea pig antiserum agai

p40.[35S]methionine-labele tractsandHSV-2-disruptec acted withguineapiganti tated proteins were separ amidegel electrophoresis,g

proteinswereexcisedand p secondpolyacrylamidegels

fixedintoplacewithmolter laidwith S.aureusV8protec

in theseconddimensionwas

B

monospecific antisera,

wehave shown that HSV-NUCLEOCAPSID 1and HSV-2 induce thesynthesis of two

molec-ular weight

classes of soluble intracellular

pro-p45

teins; these are intracellular p40 and p80, which |p40 are

immunologically

related to each other and to

polypeptides p40 and

p45,

which

are

associ-ated with

nucleocapsids isolated from cell nuclei.

Proteins

having the

mobility of intracellular p80

were

found

in

neither virions

nor

nucleocapsids.

The

finding of

a

nucleocapsid protein

sharing

antigenic determinants with

a

non-nucleocapsid

protein

was

also observed in

cells

infected with

simian

herpesvirus

SA8

(data

not

shown),

indi-cating that this

property

is

not

restricted

to

HSV. The

immunological data

suggest

that

HSV

intracellular

p40 and p80

possess common

amino

acid

sequences,

although

the

peptide

maps

of

these

protein classes showed differences in

at

least

some

of their

methionine-containing

pep-tides. The number and size of these peptides

are

dependent

on

the number and distribution of

methionine residues and accessible

proteolytic

cleavage

sites,

aswell as on

the molecular

weight

of the

protein.

Therefore, it is

not

inconsistent

that

the

peptide

map

of

intracellular p80

con-tains

fewer

methionine-containing peptides

than

the

peptide

map

of the

smaller

intracellular p40

class. The data indicate that regions of

intracel--40K -- *

lular

p40

and

p80

differ in primary structure,

whereas other

regions

possess

related amino acid

sequences.

Large and

small

T

antigens of simian

- 28K _

*virus

40 and polyoma virus contain unique and

-25K

common amino acid sequences (8, 10, 11) which

- 18K are generated by a mechanism involving

post-- 17K - _ transcriptional splicing of messenger RNA.

Elu-cidation of the mechanism

responsible for the

-14K - _

putative common amino

acid sequences and of

-11K

|distinct

peptide maps of

intracellular p40

and

p80 would require characterization of the

struc-- 8K

tural genes and the mRNA specifying these

pro-tein classes and studies

on

their

primary

struc-ture.

The

causes

for the

differences in

mobility of

P15

nucleocapsid p40

and

p45

and the

closely spaced

P4

^0

bands of

intracellular

p40

and

p80

are

probably

related

to

structural

variationsamong the

poly-NU CL EOCA PSI D

peptides

in these bands. These variations

may

be

due

to

post-translational modifications,

such

proteins precipitated

by asphosphorylation or acetylation, to proteolytic

nst

HSV-2

nuckocapsid

cleavage, and/or topossible differences in amino

lano3v-z- necrea ceut ex-dnucleocapsidswere

re-serum.After the

precipi--ated by SDS-polyacryl-,elsectionscontainingthe )lacedacrossthetopofa

-lab. The gel sectionswere

agaroseandwere

over-ase(1ug).Electrophoresis conductedatlow voltage

toallowpartialdigestioninthestackinggel,anda

fluorograph wasprepared. The gel segments used containedintracellularp40andp80 fromcellextracts

(A) and thep40 and p45proteins from disrupted nucleocapsids (B). Autoradiographs of gelsegments containing theprecipitatedproteinsservedas

refer-encesforthesecond-dimension electrophoresis step (top).80K, 80,000 molecularweight.

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A B C

p40 p80 p40 p80 p40 p80

40K

-28K- * -28K

18 K-

-17K-

_4

-17K 4fl

14K-_j_

1 1K- -1 1K ,,§W.

8iK-

-FIG. 7. Peptide maps of HSV-2 intracellular p40 and p80. Peptide mapping was performed as described in the text, except thatthe gel segments from the first electrophoresis step were oriented so that the bands were perpendiculartothe directionof the second electrophoresis step. The purified proteins were partially digested with0.1,ug(A),0.5pg(B), and1pg(C) of S. aureus V8 protease. 40 K, 40,000 molecular weight.

P156

acid sequences in portions of the polypeptide

A

chains. The

peptide

mapping

studies indicate

p12

that the

predominant polypeptide species of

in-tracellular

p40 is

closely related

to

nucleocapsid

p40 and p45.

However, intracellular p40 also

possesses

polypeptides

which

are

structurally

p40

different from

nucleocapsid

p40, since under the

same

conditions of

partial proteolytic

digestion,

the

digest of intracellular p40 contained peptides

that

were

absent in the

nucleocapsid

p40

and

p45

digests. The

reasons

for the appearance of

p15

these peptides have not yet been determined.

B

Little is known about the biochemistry of

E

herpesvirus

assembly.

Studies

employing

elec-E

tron

microscopy

indicate that

nucleocapsids

are

assembled in the nucleus and

are

then

enveloped

en

I A

I

Aduring

passage

through

the nuclear membrane

to

form complete infectious particles, which are

c

found in the

cytoplasm

(5).

Although

the

func-tion of

intracellular p80 remains

unknown,

we

believe that intracellular p40 participates in

vi-p40 rus

assembly,

since the

p40

and

p45 proteins

are

C

I

major constituents of intranuclear

nucleocap-arations immunoprecipitated by guinea pig antise-rum againstHSV-1 nucleocapsidp40. Virions and nucleocapsids werepurified from cells labeled with

p4s5

[35S]methionine

between 18 and 22 h after HSV-1 infection. The labeled virus particles (100,000 cpm) weredissociated andreacted withguinea pig anti-serumagainstHSV-I nucleocapsidp40. Proteinsof

nucleocapsids,virions, and immunoprecipitateswere

D separated by electrophoresis. Scanning densitometer

p40 tracings of autoradiograms and

fluorograms

were prepared. Autoradiogram tracings show polypeptides of HSV-1 nucleocapsids (A) and virions (B) aftera 45 13-day exposure. Fluorogram tracings show polypep-tides precipitated by guinea pig antiserum against HSV-1 nucleocapsidp40from disrupted HSV-I nu

-FIG. 8. SDS-polyacrylamide gel electrophoresis of cleocapsids aftera 13-day exposure(C) and virions polypeptides of HSV-I nucleocapsidandvirion prep- aftera40-dayexposure(D).

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[image:8.510.110.401.68.206.2] [image:8.510.57.253.262.632.2]

652 ZWEIG ET AL.

sids. The

questions

of the

presence

of

nucleocap-sid p40

and

p45

in virion

particles

and their roles in virus assembly are currently being studied in

this

laboratory.

ACKNOWLEDGMENTS

WethankM.Chakrabartyand L. Newman for excellent technical assistance and J. E. Elser and M. A. Gonda for expert assistance with electronmicroscopy.

This work was supported under Public Health Service contractN01-CO-75380 with the National Cancer Institute.

LITERATURE CITED

1. Bonner, W.M., and R. A. Lasky. 1974. A film detection method fortritium-labeledproteins and nucleic acids in polyacrylamidegels. Eur. J. Biochem. 46:83-86. 2.Bordier, C., and A. Crettol-Jarvinen. 1979. Peptide

mapping of heterogeneous protein samples. J. Biol. Chem. 254:2565-2567.

3. Cassai, E.N., M.Sarmiento,and P. G.Spear. 1975. Comparisonof the virionproteinsspecifiedby herpes simplexvirus types1and 2. J. Virol. 16:1327-1331. 4. Cleveland, D. W.,S. G. Fischer,M. W.Kirschner,

and U. K. Laemmli.1977.Peptide mapping by limited proteolysisin sodiumdodecylsulfate andanalysis by gelelectrophoresis.J.Biol. Chem. 252:1102-1106. 5.Darlington,R.W.,and H.L.Moss.1969.Theenvelope

ofherpesviruses. Prog. Med. Virol.11:16-45. 6.Gibson, W., and B.Roizman. 1972.Proteinsspecified

by herpes simplex virus. VIII. Characterization and composition ofmultiplecapsidforms ofsubtypes1and 2.J.Virol. 10:1044-1052.

7. Heilman, C. J., Jr., M. Zweig, J. R. Stephenson, and B.Hampar.1979.Isolation of a nucleocapsid polypep-tide of herpes simplex virus types 1 and 2 possessing immunologically type-specific and cross-reactive deter-minants. J. Virol.29:34-42.

8. Hutchinson, M. A., T. Hunter, and W. Eckhart. 1978. Characterization of T antigens in polyoma-infected and transformedcells. Cell 15:65-77.

9. Lostrum, M. E., M. R. Stone, M. Tam, W. N. Burnette, A. Pinter, and R. C. Nowin8ki. 1979. Monoclonal antibodiesagainst murine leukemia viruses: identifica-tion of six determinants onthe p15(E) and gp7O enve-lope proteins. Virology 98:336-350.

10.Pauca, E., A.Mellor, R. Harvey, A. E. Smith, R. M. Hewick, and M. D. Waterfield. 1978.Largeandsmall tumor antigens from simian virus 40 have identical aminoterminimapping at 0.65 map units. Proc. Natl. Acad.Sci. U.S.A. 75:2165-2169.

11. Simmons, D. T., and M. A. Martin. 1978. Common methionine-tryptic peptides near the amino-terminal end ofprimate papovavirus tumor antigens. Proc. Natl. Acad. Sci. U.S.A.75:1131-1135.

12. Spear, P.G., and B. Roizman. 1972. Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpes virion. J. Virol. 9:143-159. 13. Zweig, M.,C. J. Heilman, Jr., and B. Hampar. 1979.

Identification of disulfide-linked protein complexes in the nucleocapsids of herpes simplex virus type 2. Virol-ogy 94:442-450.

14. Zweig, M., C. J. Heilman, Jr., H. Rabin, R. F. Hopkins M,R. H.Neubauer,and B. Hampar. 1979. Produc-tionof monoclonal antibodies against nucleocapsid pro-teins of herpessimplex virus types 1 and 2. J. Virol. 32: 676-678.

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