Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus).

Vol.40,No. 2 JOURNAL OF VIROLOGY, Nov. 1981,p.367-378

0022-538X/81/110367-12$02.00/0

Proteins

Specified

by

Bovine

Herpesvirus

1

(Infectious Bovine

Rhinotracheitis Virus)

VIKIAM MISRA,* ROBERT M. BLUMENTHAL,t AND LORNE A. BABIUK

Departmentof Veterinary Microbiology, WesternCollegeof Veterinary Medicine, University of

Saskatchewan,

Saskatoon,

Received 2February 1981/Accepted 1 July 1981

An electrophoretic analysis of radioactively labeled, purified, "empty" and

DNA-containing infectious bovine rhinotracheitis virions revealed thepresence

of25 to 33 structural (virion) polypeptides. Atotal of11 of these polypeptides

could be labeled with [3H]glucosanine andwere identified as glycoproteins. In

additiontothe25structuralpolypeptides, infectious bovine rhinotracheitis

virus-infected cells also contained atleast 15 nonstructural (nonvirion) polypeptides

thatwerenotpresentinpurified virions. Expression of the viral polypeptides in

infected cellswascontrolled temporally. Thus,mostviralpolypeptidescould be

categorized as "a" (immediate early), ",B" (early), or"y" (late) on the basis of

their order of appearance in infected cells and whether their syntheses were

dependentuponprior viralproteinorDNAsynthesis.Noneof theglycoproteins

belonged tothe a class, although atleast one (GVP11) wassynthesized in the

absence ofviral DNA synthesis. Serum from a cow in which infectiousbovine

rhinotracheitis virus lesionswerereactivated by dexamethasoneprecipitatedboth

structuralandnonstructuralpolypeptides.

Bovineherpesvirus 1,whichis also knownas

infectious bovine rhinotracheitis virus (IBRV),

is similarinstructuretootherherpesvirusesand

possessesalineardouble-strandedDNA genome

which hasamolecularweightofapproximately

10 (J. E.Farley,I. B.Skare,andJ.Skare, Abstr.

Int.Conf.HumanHerpesviruses, 1980).Like the

genomesof otherherpesviruses, thegenome of

IBRV is comprised of L and S components,

althoughonlytwogenomicisomers exist in

prep-aration sinceonlytheScomponentispresentin

both of thetwopossible orientations(Farleyet

al., Abstr. Int. Conf. Human Herpesviruses,

1980). Also like otherherpesviruses, IBRVcan

remain latentinanimals, probablyintrigeminal

ganglions,and can be reactivated with relative

ease(12,21). IBRV isanimportantpathogen of

cattle and can cause severe respiratory

infec-tions, vulvovaginitis, abortions, conjunctivitis, meningoencephalitis, and generalized systemic

infections (9). Thus, IBRV represents a good

modelforstudyingthebiologyandimmunology

of active and latent herpesvirus infections in

natural hosts.

Asmany as 33 virionpolypeptides havebeen

identifiedfor other herpesviruses (4-6, 11,

27-29). In herpesviruses 1 and 2, at least five of these structural polypeptides are glycoproteins.

t Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor,NY11724.

Theseglycoproteinsarealsopresent onthe

sur-faces ofinfectedcells,andatleastsomeof them

areinvolved in immunerecognitionand immune

cytolysis (18).

The herpesvirus proteins can be designated

a, ,B,and-y,dependingonthetemporalorder of synthesis and whether synthesis is dependent

uponthesuccessfulprogressionof certain phys-iological processes in infected cells (24). The transcriptionofa orimmediateearlygenesdoes

notrequire theexpressionof other viral genes,

and in the absence of protein synthesis (e.g.,

after treatment with cycloheximide) mRNA's

from a genes accumulate. If the protein

syn-thetic blockisremoved,aproteinsareproduced

inlargeamounts evenin the absenceof further

mRNA synthesis (24). The synthesis of /3 or

early proteinsisdependentupontheprior

syn-thesis of theaproteinsand inturnleadstothe

cessation ofaproteinsynthesis.

'The

transcrip-tionofy orlategenesrequirestheprior

expres-sionofa and

/B

viralDNAsynthesis.

There are considerable data available

con-cerning thekineticsof virusreplicationinvitro

andimmunedestructionofIBRV-infected cells

by bovine leukocytes (1, 25). Very little

infor-mationhas beenreported concerningthe

corre-lation of immune destruction to specific

poly-peptide synthesis and expression on host cell

membranes. Inanattempttoidentifythe poly-367

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368 MISRA, BLUMENTHAL, AND BABIUK

peptides involved in immune destruction, the

polypeptides expressed in latently infected cells,

and the temporalpatternof polypeptide

synthe-sis in lytically infected cells, we tried to identify

and characterize allof the polypeptides

synthe-sized in IBRV-infected cells. Here we describe

the identification of more than 25 structural

polypeptides, 11of whichareglycoproteins and

15ofwhicharenonstructural polypeptides. We

also attempted to assign the IBRV-specified

polypeptidestothea,,B,and y temporal classes.

MATERIALS AND METHODS

Virusand cells. Strain P8-2 of IBRVwascultured

inGeorgia bovinekidneyorMadin-Darbybovine

kid-neycells as describedpreviously(1).Briefly,cells were

grown toconfluency inpetridishes (diameter, 60 or

100mm;Corning Glass Works, Corning, N.Y.) in Eagle

minimal essential medium(MEM) supplementedwith

5%fetal bovineserum.

Infection and labeling of cells. Monolayers of

bovine kidney cells were infected with IBRV at a

multiplicity of infection of10PFU per cell.After the

virusabsorbed for1 hat370C,themonolayerswere

washed with MEM andoverlaid with methionine-free

MEM (catalog no. 79-0115; GIBCO Laboratories,

GrandIsland, N.Y.) containing2%fetal bovine serum.

After incubation for6h to permit cessation of host

protein synthesis, 25,LCiof[35S]methionine (catalog

no. SJ204; Amersham Corp., ArlingtonHeights, Ill.)

permlwasaddedtoeach tissue culture dish. The cells

wereharvested20h afterinfection. To label

glycopro-teins, 25,ICiof[3H]glucosamine(catalogno.TRK398;

Amersham)permlwasaddedtothe infected cultures

6hafterinfection.

Toidentify proteins synthesized in the absence of

DNA synthesis, cells were infected and then

main-tained in the presence of50,ugofcytosinearabinoside

(ara-C;Cytosar;TheUpjohn Co., Kalamazoo, Mich.)

perml. At6hafterinfection, the mediumwasreplaced

with5mlof MEMcontaining50,uCiof

[3S]methio-nine per ml and 50,ugof ara-C per ml.

Toidentifyapeptides,cellswereinfected and

main-tained in the presence of50,ugofcycloheximide

(Boeh-ringer MannheimCorp.,NewYork, N.Y.)perml. At

6hafterinfection,thecellswerewashedextensively

with MEM before the addition of MEMcontaining50

gCiof[3S]methionineperml and 2.5,ugof

actinomy-cinD(Sigma Chemical Co., St. Louis, Mo.) per ml.

Purification of virus. [3S]methionine- or

[3H]glucosamine-labeled virus waspurified from the

growth supernatant of infected cells. After the cell

debriswasremovedby centrifugationat 500xgfor

10 min, the virus was pelleted by centrifugation at

100,000xgfor2h. The viruspelletwassuspendedin

1mlofphosphate-buffered saline (PBS) (0.15MNaCl,

2.5mMKCI, 1.5mMKH2PO4,8mMNa2PO4,0.02%

dextrose,pH 7.4) by repeated pipetting and mild

son-ication (1to2 s at alow powersetting). The viruswas

thenlayeredonto an11-ml linear20to50%potassium

tartrategradientin 0.15 MNaCl-0.01MTris-0.001 M

EDTA(pH 7.5) andcentrifugedat80,000xg for1.5

h at 4°C. Fractions (0.2ml) were collected,and the

J. VIROL.

virus-containingfractionswereidentifiedby

measur-ing theradioactivity in each fraction. These fractions

werethenpooledanddiluted withPBS,and the virus

wasrecoveredbypelleting.

Preparation of cell extracts. The cell extracts werepreparedby the method of Purifoy and Powell

(22).Approximately 107 pelleted cellsweresuspended

in1ml ofasolutioncontaining20mMTris(pH 7.5),

2mM,B-mercaptoethanol,and 500 g of bovineserum

albumin per ml. After thispreparationwassonicated

for 2 min at apowersetting of7 in a Sonifier cell

disrupter (Biosonics, Plainview, N.Y.), 1ml of1.7M

NaCl-5 mM EDTAwasadded,andthe mixturekept

on ice for 40 min.The resulting precipitate was

re-movedbycentrifugation at30,000 xgfor20minat

4°C, and the mixturewasdialyzed againstasolution

containing200mMTris(pH7.5),50mMNaCl,1mM

EDTA, 2mM/i-mercaptoethanol, and 10%glycerol.

Thelight precipitatethat formedduring dialysiswas

removedbycentrifugationat100,000xgfor60minat

40C.

PreparationofStaphylococcusaureusstrain A

adsorbant. The Cowan strain of S. aureus was

ob-tained from the American Type Culture Collection

(ATCC12598) andwaspreparedforadsorption by the

technique of Kessler (13). The bacteriaweregrownfor

24 h at 37°C per ml in brain heart infusion broth

(GIBCO)supplementedwith4,ugofniacin per ml and 2Mugofthiamine-hydrochloride perml;then the cells

were harvested by centrifugation and washed twice

with PBS-2 (150 mM NaCl,20mMNa2HPO4, 20 mM

NaH2PO4, pH 7.2) containing 0.05% sodium azide.

After thecellsweresuspendedto aconcentrationof

10% (vol/vol) in PBS-2 containing azide, they were

stirredat roomtemperaturefor1.5h inthe presence

of1.5%Formalin, washed,andsuspendedtothesame concentration in buffer without Formalin. The cells

were then stirred for 5 min at 80°C, and this was

followed by rapid cooling on ice. After two washes

with PBS-2containing azide, the bacteria were

sus-pended to afinal concentration of 6% (vol/vol) and

storedat4°C.Before adsorption,the cellswere

pel-leted, suspended to a concentration of 10% in 0.5%

Nonidet P-40(Shell)in NETbuffer(150mMNaCl,5

mM EDTA, 50mM Tris, pH 7.4, 0.02% azide),and

incubatedat roomtemperaturefor15min.They were

then washed once and resuspended in fresh buffer

containing0.5% Nonidet P-40tothesamevolume. Forimmunoprecipitation,the bovine anti-IBRV

im-munoglobulin G fraction and the cell extract were

incubated inoptimumproportions for1h at37°C; this

wasfollowed by the addition of200

pl

cellsuspension.After thecells stood for90minat4°C

and were washed once with NETbuffer containing

Nonidet P-40, they were suspended in 50

pl

sodiumdodecyl sulfate (SDS)-6 M urea, incubated for

15 min at 45°C, heated at 100°C for 3 min, and

analyzed bySDS-polyacrylamide gel electrophoresis

(PAGE).

Analysis ofproteins by PAGE. Samples were

electrophoresed in the presence of SDSthrough 7.5

and 10%polyacrylamide gels (15) by using a 15-cm

verticalelectrophoresis apparatus (Richter Scientific,

Vancouver, Canada). Gels containing'S-labeled

sam-pleswere dried and autoradiographed on Kodax

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VOL. 40, 1981

OMAT-R film. 'H- and 14C-containing gels were

soaked in En3Hance (New England Nuclear Corp.,

Lachine, Canada) and fluorographed on preflashed

film.

The molecular weights of the radioactive bands

weredetermined by comparing theRfvalues of these

bands with theRfvalues of markers of known

molec-ularweights(high-andlow-molecular-weight

calibra-tion kits; Pharmacia, Uppsala, Sweden)

electropho-resed on the samegel. Molecular weight markerswere

visualized by staining the gels with Coomassie blue

beforedrying.

Two-dimensional gel electrophoresis. The

technique of O'Farrell (19) was used, with the

modifi-cations described by Garrels (8). Virus pellets were

suspended in

100-pd

0.24% SDS and 1.4M /3-mercaptoethanol; 2 ul of0.5

M Tris(pH8.8)buffercontaining1mgof DNaseI,0.5

mg of RNase A per ml, and 0.1 M MgCl2 was then

added. After thepreparationstoodonice for5 min,

100 mgof solidurea wasadded, followedby15ml of

lysis buffer (10 M urea, 4% Nonidet P-40, 0.5 mM

lysine-hydrochloride,0.1Mdithiothreitol, 0.25% SDS,

4.5%glycerol, 0.05 ml of ampholytes per ml).

Polypep-tideswereseparated along the first dimensioningels

PROTEINS SPECIFIED BY IBRV 369

containing9partsofBiolyte (pH5to7)and1partof

Biolyte (pH3to10) andalong the second dimension

bySDS-PAGEon10%gels. The gelswere thensoaked

inEn3Hance (NewEnglandNuclear), dried,and

fluo-rographed.

RESULTS

Purificationof IBRV. Theaccurateanalysis

of virionproteins requires thatthevirus

prepa-ration be essentially free of cellular and

non-structuralviral proteins. Potassium tartrate

gra-dients wereusedtopurify IBRV since they have been used to purify other herpesviruses (5, 14) and give good yields ofpure virus. To reduce

contamination with host cell proteins further,

the virus was purified from the extracellular

medium rather than fromacelllysate.

The efficacy of potassium tartrate gradients

in the purification of IBRV was evaluated by

labeling the purifying virus (Fig. 1). At every

stage of the purification process, samples were

treated with trichloroacetic acid, and the

amounts of 3H-labeled (viral) protein and

14C-Prelabel bovine kidneycells with 14C-amino acids

I 20h(labeling)

Wash, add nonradioactive MEM

l 2h (chase)

Infect withIBRV

J 6 h (toallow virus-induced cessation of hostprotein

synthesis)

Add3H-amino acids

I 24 h(labeling)

Harvestcells

Centrifuge 15min

(500xg) /

Celldebris Supernatant

Centrifuge 2h

(100,000xg)

Pellet, resuspend Supernatant

inPBS

Firstpotassiumtartrate 90min

gradient

(80,000xg)

Fractionate, pool viral peak,

dialyze Secondpotassiumtartrate 90min

gradient

(80,000xg)

Fractionate, pool

viralpeak

FIG. 1. Evaluationofpotassiumtartrategradientsas ameansofpurifyingIBRV.

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370 MISRA, BLUMENTHAL, AND BABIUK

labeled (cellular) proteinweredetermined.

Ta-ble1showsthatsimplecentrifugation at 100,000

x g resulted in significant purification since

morethan90% of the'4Cremained inthe

super-natant, whereas almost all of the 3H (added to

the cell culturesafter cessation of host protein

synthesis) wasremoved inthe pellet. Through

thenext twostepsofpurification, the ratio of 3H

to 14C remained relatively constant, although

therewasaconsiderableloss oftotal

radioactiv-ity. In subsequent experiments the virus was

centrifugedthrough onlyonetartrategradient.

To determine whether the small amount of

14C that remained associated with the virus in

the experiments described above was due to a

breakdown of cellular proteinsfollowed by

rein-corporation of the released label into viral

pro-teins or due tocontamination by cellproteins,

we performed the following experiment. Cells

were grown for 20 h in the presence of

[tS]-methionine, washed,grownforanadditional2h

in isotope-free MEM, and then infected with

IBRV. No radioactiveprecursor wasadded after

infection. Virus was purified from the cell-free

growth supernatantasdescribed above. The

vis-ible virus band was recovered from the first

tartrate gradient, pelleted, and analyzed by

SDS-PAGE along with labeled IBRV marker

proteins. The smallamountofradioactivity

as-sociated with the viruswasdistributedevenlyin

all viral polypeptides, suggesting that amino

acids fromprelabeled cellproteinswere

proba-bly released andreincorporated. In any case,it

seemed clear that thepurification protocol

de-scribed above yielded IBRV virions that were

essentiallyfree of host cellproteins.Virus

puri-fied in thismanner was also free of

contamina-tion withnonstructuralviralproteins since

non-TABLE 1. Purification ofIBRV6

Trichloroacetic acid-precipitable Sample radioactivity (cpm)

3H 14C

Cell-freegrowthsuper- 4.7 x106(100)b 9.2 x104(100) natant

100,000-x-gsuperna- 1.7x105(36) 8.6 x104(93) tant

100,000-x-gpellet 4.9 x106(100) 3.0 x104(32)

Virus bandafterfirst 1.42 x106(30) 1 xl04(10)

tartrategradient

Virus band aftersecond 2.5 x105(4.3) 8.6 x102(0.9)

tartrategradient

'Georgiabovinekidney cellswerelabeledwith"4C-amino

acids before infectionandwith3H-amino acidsafter infection

with IBRV.Virus was purifiedfromgrowthsupernatantfrom

which the cell debris hadbeenremovedbycentrifugationat 500xgfor15min.

bNumbersinparentheses arepercentages ofthetrichlr roaceticacid-precipitable radioactivityin thecell-freegrowth

supernatant.

J. VIROL.

structural viral peptidespresentin theinfected

cells inrelativelyhigh concentrations werenot

apparent in the virus preparation even after

prolonged autoradiography ofthe gel (data not

shown).

IBRV structural proteins. In potassium

tartrate gradients IBRVwasresolved intotwo

distinctpeaks (Fig. 2A). [3S]methionine-labeled

IBRVwasrecovered from the radioactivepeaks

andanalyzed bySDS-PAGE,followedby

auto-radiography andfluorography.Thegelsrevealed

25 bands, which were designated GVP1, VP2,

GVP3, VP4, GVP5, GVP6, GVP7, VP8, GVP9,

VP10, GVP11, VP12, VP13, VP14, GVP15, GVP16, VP17, VP18, VP19, GVP20, GVP21, VP22, VP23, VP24, and VP25. These bands

*0

CE)

x

U)

0

±

(l.)

FRACTION#

FIG. 2. Analysis ofIBRV onpotassium tartrate

gradients.(A)[3Slmethionine-labeledIBRVwas

re-coveredfrom thegrowth supernatant by

centrifuga-tion. Thepelletwassuspended in1 mlofPBS and

layeredonto a 20 to50%potassiumtartrategradient.

Aftercentrifugation for90minat80,000xg, 0.2-mi

fractionswerecollected, and theradioactivity

asso-ciated with eachfractionwasdetermined.Fractions

14 to 20 were combined intopool I (peak I), and

fractions22 to 26 were combined intopoolII(peak

II). Viruses were recovered from thesepools and

analyzedby PAGE (see Fig. 3). (B)Infectedcellswere

labeled with either [3H]thymidine or "C-amino

acids.At 24 hafter infection.th-c,, uwthsupernatants

were combined, o"I ahe virus was recovered and

analY7pAJ . a20to50%potassiumtartrategradient.

2,actions12to40 wereprecipitated with

trichloro-acetic acid, and the amountsof"C and 3H in the

macromoleculesofeachfractionweredetermined.

I

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PROTEINS SPECIFIED BY IBRV 371

ranged in molecular weight from more than

330,000 toless than 14,000(Fig.3and Table2).

A total of 11 of these bands (GVP1, GVP3,

GVP5, GVP6, GVP7, GVP9, GVP11, GVP15,

GVP16,GVP20,andGVP21)wereglycoproteins

since they were labeled with

[3H]glucosamine

(Fig.4). GVP9 and GVPllwere presentin

rel-atively large amounts. Some of the remaining

bands were lighter and were visible only after

prolonged exposure. GVP9, GVP5, GVP6, and

GVP15 either hadverylow methioninecontents

or wererelatively highlyglycosylatedsincethey

were not labeledextensively with

[3S]methio-nine.

Peak II, the more slowly sedimenting peak,

differed from peak I in that it lacked VP17,

VP22, and VP24, but the glycoproteins were

present atroughlythesamelevelsin bothpeaks.

3 - 1413 15 4417 418 419 2 0 422 M423 424 .425

FIG. 3. Analysis of

r35SJmethionine-labeled

by electrophoresison a10%polyacrylamidegel.

Vi-ruses were recovered from peaks I and II of the

potassium tartrategradient shown in Fig. 2A. The

open arrowheads indicate viralglycopeptides, and

the solid arrowheads indicate

non-glycosylatedpoly-peptides. Viralpolyl GVP VP2 NSa GVP NSb NSc VP4 GVP GVP NSd NSe GVP' NSf VP8 NSg NSh NSi GVP vPl1 GVP VP12 VP13 VP14 NSJ NSk GVP GVP VP17 VP1E VP19 GVP NS1 GVP NSm NSn VP22 NSo VP23 VP24 VP25

TABLE 2. Viralpolpeptidesa

Mol wt cal- Mol wt

cal-culated from cal-culated firom

peptide 10% gel 7.5% gel

(X103) (X103) 1 >330 263 185 -3 182 182 166 141 5 138 6 130 126 120

7 104.7 105

93

89.1 91

89.1 91

81.2 87

83

9 77.6 82

74.1 79

'11 67.6 74

63.9 69

ib 63.4 65

1 62.9 62

58.2 60

56.1 57

'15 54.9 55

16 53.9 54

3b 20 21 52.8 50.9 50.0 49.1 48.2 42.2 38.4 36.3 34.8 29.4 26.2 16.2 13.9 48 41 39 35 32 Cate-gory Y 9 9 Y a a ?

11

aPolypeptideswerenumberedinorder of

decreas-ing molecularweight. Structural polypeptides were

designated VP (virionpolypeptide)orGVP

(glycosyl-ated virionpolypeptide),and nonstructural

polypep-tides were designated NS andwere assigned

lower-caseletters.

bThesespeciesmay becomposedofmorethanone

polypeptide since they produced multiple spots on

two-dimensionalelectrophoresisgels.

Therefore,it isunlikelythatpeakIIrepresented

nucleocapsids and peak I contained complete

particles.To determine the nature of these two

IBRV peaks, infected cells were labeled with

either [3H]thymidine or

"4C-amino

([3H]-thymidine and

"4C-amino

and proteins, respectively). When most of the

cells had detached from thegrowthsurface,the

VOL. 40,1981

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372 MISRA, BLUMENTHAL, AND BABIUK

FIG. 4. Analysis ofpurified[aH]glucosamine-and

[35S]methionine-labeled IBRV. 3H- and 35S-labeled

viruseswerepurifiedonpotassiumtartrategradients andanalyzed by electrophoresison10%(lanes 1 and

2) and 7.5%(lanes3through 5)polyacrylamidegels. Lanes 1, 3, and5, [3H]glucosamine-labeled IBRV (theselanescontaineddifferentamountsof radioac-tivity andwereexposed for varying lengths oftime to

illustrate all radioactive bandsmoreclearly); Lanes

2and4,[35S]methionine-labeledIBRV.

culture supernatantsweremixed, and the virus

was pelleted and then centrifuged through a

potassium tartrategradientasdescribed above.

Macromoleculesinfractionsof thegradientwere

precipitated with trichloroacetic acid, the

pre-cipitatesweretrappedonglassfiberfilters, and

the amounts of 3H and 14Cineachfractionwere

determined. AsFig.2Bshows,peakIcontained

both 3H and '4Candprobably represented

com-pleteparticles. Ontheotherhand, peakII

con-tainedonly '4C andwasprobably comprised of

particlesthat lackedDNA.

Analysisof IBRV structural proteins by two-dimensionalgelelectrophoresis.To

re-solve IBRV structuralproteinsfurther, weused

two-dimensionalelectrophoresistoseparate

pro-teinsalongonedimensiononthebasisofcharge

differences by isoelectric focusing and along the second dimension on thebasis of size on

SDS-polyacrylamide gels (19). This technique has

been used recently to resolve vaccinia virus

structuralpolypeptidesintomorethan100spots

(7)and herpes simplex virus1-infectedcell

poly-peptides into200spots (L. Haarr and H.

Mars-den, Abstr. 4thCold SpringHarborSymp.

Her-pesviruses, p. 109,1979).

Figure 5 shows theresolutionof IBRV

struc-turalpolypeptidesontwo-dimensionalgels.The

approximatepositionsof the IBRVpolypeptides

separated by SDS-PAGE alone are indicated.

Thisfigure alsoshowsthepositions of the spots

that consistently appeared when the analysis

wasrepeated. Electrophoretic streaking was

al-waysobserved for thepolypeptides which were

probably VP4 and VP8. Similar heterogeneity

has also been observed with the 60,000- and

62,000-dalton major virion peptides of vaccinia

virus (7) and the phosphorylated major coat

protein (VP1) of simian virus40(20).Spotsthat

possessed electrophoretic mobilities through

10% SDS-acrylamide gels which were

compa-rable to the mobilities ofglycoproteins GVP1,

GVP5, GVP6, GVP7, and GVP16 appeared as

multiplespots.

Infected cell proteins. Bovine kidney cells

were infected with IBRV at a multiplicity of infection of10PFU/cell.At 6hafterinfection,

[35S]methionine

an-alyzed bySDS-PAGEon10and 7.5%

polyacryl-amidegels(Fig. 6A and B, lanes 2). The analysis of the viral proteinsin infected cellswas made easier because withamultiplicityofinfection of

5 to 10 PFU/cell, by6h afterinfection all host

proteinsynthesiswasgreatlyreduced and there-fore theradioactive amino acid precursors that

were added at that time were incorporated

al-most exclusively into viral proteins. As Fig. 6

shows, the bands in the infected cell sample

(lanes2) didnot have thesameelectrophoretic

mobilitiesasthe bands in the mock-infected cell

sample (lanes 1). Even the dark 46,000-dalton

host bandwas almost completely missing from lanes 2. In additionto containing the 25 IBRV structural polypeptides, the infected cells also contained12nonstructuralproteins,whichwere

designated NSc, NSd, NSe, NSf, NSg, NSh,

NSj, NSk, NSl, NSm, NSn, and NSo. These

polypeptides ranged in molecular weight from

182,000to 32,000(Table 2).

Todetermine thetemporalorder ofsynthesis

of thesestructuralandnonstructuralproteinsin

infectedcells, bovinekidneycellswere infected with IBRV andlabeled with[35S]methioninefor

2-h periods at 0, 2, 4, 6, 10, and 12 h after

infection. Mock-infected cellswerelabeled for2 J. VIROL.

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PROTEINS SPECIFIED BY IBRV

A

+ .w

,I< <7

48

:* 4,13

41.

<116

422

425

FIG. 5.Analysis of[3Slmethionine-labeledIBRVby two-dimensionalelectrophoresis.Thepolypeptides of

[3S]methionine-labeledIBRVwereseparated alongthehorizontal axisbyisoelectricfocusingandalongthe

vertical axisonthe basisofsizebyPAGE. A and B indicate the acidic and basic endsofthefirstdimension

(approximately pH3.5 and8.5,respectively).Thearrowsindicate spots and streaks thatconsistentlyappeared

whendifferent preparationswereanalyzed. Thearrowheadsontherightindicate theapproximate positions

oftheIBRVpolypeptidesseparated byone-dimensionalelectrophoresis; glycopeptidesareindicatedbyopen

arrowheads.

hatthe timeof mock infection. The cells were

harvestedatthe endofthelabelingperiodand wereanalyzed by SDS-PAGE(Fig. 7). No

radio-active viral polypeptides were observed inthe

first 2 h afterinfection, probably because viral proteins at this early stage were produced in

insufficient quantitiestobeobservedagainst the heavy background ofhost polypeptides. How-ever,between2 and 4 h afterinfection, polypep-tides with electrophoretic mobilities similar to

those of VP4, NSb, NSc, NSd, NSi, and NSj

were observed against a slightly less intense

background of hostproteins. Between4and 6 h afterinfectionGVP7, VP8, NSf, NSl, NSm, and

NSnwerealso synthesized.Inaddition, the

in-tensity ofpeptide NSj beganto decrease. The

remaining viral polypeptides were labeled

be-tween6 and 8 h afterinfection.Almostallofthe

viral polypeptides that were labeled in the

2-and 4-hlabelingperiodswerealsolabeledat6h, althoughthe intensities of bands NSdand NSi decreasedandNSjdisappeared.Inaddition,

ces-sation of host polypeptide synthesis, as

deter-minedbythealmostcomplete absence of

radio-activity in the46,000-dalton host band, occurred

at6 hpostinfection.

Thepattemoflabeling remained unchanged

in the 10- and 12-hlabelingperiods.

Herpesviruses are generally believedto

pos-sess acomplexsystem ofgeneregulation,where

expression ofyorlategenesrequires prior syn-thesisofaand,B proteins,aswellastheonsetof

viralDNAreplication (16, 29, 30). Therefore,we

examined the pattern of protein synthesis in

IBRV-infected cells in which DNA synthesis had been inhibited bytreatment with 50,ugof

ara-Cperml. This concentration was 10 times

thatrequiredtoshut down viral DNAsynthesis in infected cells almost completely (data not

shown).

Immediate early (a), early (8), and late (y)polypeptides. Toidentifyimmediateearly, early, and lateviralproteins,bovinekidneycells

weremock-infectedorinfectedwithIBRV; then

these cells were incubated in the presence of

either50,ugofcycloheximide permlor50,ug of ara-Cperml, ortheywereleftuntreated. At 6

h afterinfection, the cycloheximide-treated

cul-tureswere washed extensively with MEM, and

the growthmediumwasreplacedwith medium

lackingcycloheximide.FurthermRNAsynthesis inonebatch ofcultureswasprevented by adding

VOL. 40, 1981

B

373

'*

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374 MISRA, BLUMENTHAL, AND BABIUK

9 8 7 6 5

t 2

a.

4,

.;

_ ..._{._}

1

i:>

F5F: 1

2 a 3.b

-4

-d -e -7 _f

-8.9

-h

-9.1

-10

-11

-12 13 -14

-i

-k

-16

Bmw9 8 7 6 5 4 3

7

I4

aa|~

-'

60* ei

480

36* 4 4

30* _ 0

-I2 1-3,b

de

-7

-8 :~1o

-11

.-1213

-i

-k -16

1--m

.n

* 22

0

.. 0

I

- -m

f -n

-2

'l-

20*

4*.

14.4*

FIG. 6. Analysis ofIrSlmethionine-labeledmock-infectedandIBRV-infected ceU proteinson7.5 and10%

polyacrylamide gels.Lanes1,Mock-infectedbovinekidney cells; lanes2,IBRV-infectedcellslabeled 6to20

h after infection;lane3,purified IBRV;lanes4,mock-infectedcellstreated with50pgofara-Cpermland

labeled 6to20 hpostinfection;lanes5,IBR V-infectedcellstreated with 50pgof ara-Cperml;lanes 6,

mock-infectedcells labeledafterreleasefrom50pgof cycloheximideperml;lanes 7, IBRV-infectedcellslabeled

after releasefrom cycloheximide; lanes8, mock-infected cells labeled in a medium containing 25 pg of

actinomycin Dpermlafterreleasefromcycloheximide; lanes9, IBRV-infectedcells labeledinamedium

containing actinomycinDafterreleasefromcycloheximide.Inlane 5 theopencirclesindicatepolypeptides

thateitherwerenotsynthesizedor weresynthesizedinreducedamountsin thepresenceofara-C. Thearrows

inleft marginindicatethepositions ofmolecularweight (MW)markers.Viralpeptidesandglycosylatedviral

polypeptidesarenumbered,andnonstructuralpolypeptidesaredesignatedwith lower-caseletters.

2.5 ,ug of actinomycin D per ml of medium.

[3S]methioninewasaddedtoallcultures. At 20

h afterinfection, the cells were harvested, and

theproteinswereanalyzedbySDS-PAGE.

yPolypeptides. Althoughmost of theviral

structuraland nonstructuralpolypeptideswere

synthesizedininfectedcells treated withara-C,

atleast five polypeptides (GVP1, GVP3, NSe, VP8, and VP13) were missing or were

synthe-sizedingreatlyreduced amounts (Fig. 6B,lane

5). Thus, thesewerethetrue 'yorlate

polypep-tides since they were not synthesized in the

absence of viral DNA synthesis. ara-C-treated cells contained an additional band, designated

NSi, which was not observed inuntreated

in-fected cells.NSimayhaverepresenteda precur-sor that was not processed in the absence of

DNAsynthesis, oritmayhave been a,

poly-peptide which was not observed in untreated

cellslabeled 6 h after infection because its syn-thesiswas inhibitedstrongly by they

polypep-tides.

aPolypeptides. Threeproteins appearedto

besynthesizedinlargeamountsafter the cyclo-heximide was removed if actinomycin D was

addedtothewashed cultures. In the absence of

actinomycin D,all of the viralpolypeptides

ob-served in the ara-C-treated cellswere

synthe-A

220 *

-24

::tg

-25

.

J. VIROL.

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PROTEINS SPECIFIED BY IBRV 375

[3H]glucosamine and analyzed by SDS-PAGE

(Fig. 8).No radioactive bandswereobserved in

the cells released from thecycloheximide block,

andonlyoneglycopeptide (GVP11)was

synthe-sized inara-C-treated cells.

/3 Polypeptides. All viral polypeptides

syn-thesized in the absence ofviral DNA synthesis

(50

jig

poly-peptides (Fig.6,lane5,and Table 2).

FIG. 7. Time courseofIBRVpolypeptide

synthe-sis. IBRV-infectedceUs werelabeled with

[9S]me-thionineat0 to2(lane 2),2 to 4(lane3),4 to6(lane

4),6to 8(lane 5),10 to 12(lane6), and12to 14(lane

7) hafter infection.Mock-infectedcellswerelabeled 0 to2hafter mock-infection (lane1).Atthe endof

thelabeling period,thecellswereharvested,andthe

proteins were analyzed by electrophoresis on 7.5%

polyacrylamidegels.

sized(Fig.6A andB, lanes 7). Thiswasprobably

because in the absence of actinomycin D, /3

transcriptsweresynthesizedand translated after

initialsynthesis ofa peptides; /8geneproducts

theninhibitedfurther translation ofamessages.

Threeapolypeptideswereidentifiedininfected

cellsafter thecycloheximide wasremoved, and

these weredesignatedNSb, NSc,andNSg (Fig.

6B, lane 9).Noneoftheviralglycoproteins could

beidentified asa proteins,and theonly

glyco-protein made in appreciable amounts in the

absence ofviralDNAsynthesisappearedtobe

GVP11. These observations were confirmed

when infectedcellsthat were treated withara-C

or released from the cycloheximide block and treated with actinomycin D were labeled with

FIG. 8. a, ,B, and y viral glycopeptides.

Mock-in-fected or IBRV-infected cells were treated as

de-scribed in the testand in thelegendtoFig. 6. The

cellswerethenlabeled with[3H]glucosamine. At20

h postinfection the cells were harvested, and the

proteins were analyzed by electrophoresis on 7.5%

acrylamidegels.Radioactivebandswerevisualized

by fluorography.LaneA,Mock-infectedcells; lane B, IBRV-infected cells; lane C, mock-infected cells

treated with ara-C; lane D, IBRV-infected cells

treated withara-C; laneE, mock-infected cells

la-beled in a medium containing actinomycin D after

releasefrom cycloheximide; lane F, IBRV-infected

cells labeledinamediumcontaining actinomycinD

after releasefrom cycloheximide. Thenumbered

ar-rowheads indicate viralglycopeptides.

VOL. 40,1981

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376 MISRA, BLUMENTHAL, AND BABIUK

Immunoprecipitation of infectedcell

pro-teins. To determine whether animals with

re-currentIBRV infections had antibodies against

structural and nonstructural IBRV

polypep-tides, immunoglobulinwaspurified fromacow

thathad been infected intranasallywith IBRV.

Recurrent IBRV lesions were induced in this

animal by administering dexamethasone (21).

The immunoglobulin preparation had a serum

neutralization titerof 1:1,000. Cell-free extracts

from infected or uninfected cells were mixed

with theimmunoglobulin preparation in optimal

proportions, and the antigen-antibody

com-plexeswerethenprecipitated with S.aureus and

analyzed bySDS-PAGE. With the exception of

a small amount of actin (molecular weight,

46,000), the antibodies did not precipitate

sig-nificant amountsofanymock-infectedcell

pro-teins.On the other hand,all

'S-labeled

polypep-tides observed in infected cells appeared tobe

precipitated (Fig. 9).

DISCUSSION

Electrophoresis ofIBRV virions on 7.5 and

10% SDS-polyacrylamide gels which resolved

polypeptides with molecular weights ranging

from 12,000 to 330,000 revealed 25 distinct

J. VIROL.

bands.Fiveof these bandsmayhave contained

two orthreeproteins,asrevealedby

two-dimen-sional electrophoresis (isoelectric focusing and

SDS-PAGE), although some ofthese multiple

spots wereprobablyartifactualinorigin(Fig. 5).

Thus,IBRVvirions containatleast25

polypep-tides, andmorelikely28to33polypeptides.This

is in goodagreementwithstudies of other

her-pesviruses; 33 polypeptideshave beenfoundin

herpes simplexvirus (28),20polypeptideshave

been foundin pseudorabies virus (29),26

poly-peptides have been found in murine

cytomega-lovirus (5, 13), and 33 polypeptides have been

found inEpstein-Barr virus (6). Of the33herpes

simplex virion proteins, 5 are glycosylated (2,

18), whereas 11 of the 28 to 33 IBRV virion

proteinscould be labeled with[3H]glucosamine

andare thought to beglycoproteins. Although

11 of28 to 33 seemshigh compared with5 of

33,theputative glycoproteins arewell resolved

inmolecularweight, andmostgive characteristic

patterns on two-dimensional gels. However, it

remainstoberigorously demonstrated thatall

11 are actually distinct glycoproteins. On

two-dimensional gels, spots that possessed

electro-phoretic mobilitiesthrough 10%

SDS-acrylam-ide gels comparable to those of glycoproteins

GVP1, GVP5, GVP6, GVP7, and GVP16

ap-__'''''

.

1

.. ....11

4iTX

1.. ... ~~~~~~~~~...

-...i.... ,XF .

FIG. 9. Immunoprecipitation of mock-infectedorIBRV-infectedcellextractswithimmunoglobulin from

cowswithreactivated IBRV lesions. Cellextractsfrom mock-infectedandIBRV-infectedbovinekidneycells

wereimmunoprecipitatedwiththeimmunoglobulin fraction fromserumobtainedfromacowwith reactivated

IBRV lesions(19). S.aureusstrain Aadsorbantwasusedtofacilitate immunoprecipitation.

Immunoprecip-itateswerewashed andelectrophoresedon7.5%polyacrylamide gels,and theradioactive bandswerelocated

byautoradiography.AutoradiographswerescannedwithaHelenaQuickScan Jr.densitometer. (A)

IBRV-infectedcells,whole-cellextract.(B)IBRV-infected cells, immunoprecipitate. (C) Mock-infected Madin-Darby

bovinekidneycells, whole-cellextract.(D) Mock-infected Madin-Darbybovinekidneycells,

immunoprecipi-tate.

F F

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VOL. 40, 1981

peared as multiple spots. Each set ofmultiple

spots may representmolecules of thesame

gly-coprotein that differ in the extent of

glycosyla-tion. Similar multiple glycoprotein spots were

observed inherpessimplexvirus1-infectedcells

by Haarr and Marsden (Abstr.4th ColdSpring

Harbor Meet.Herpesviruses, p. 109, 1979) who

showed bypulse-chase experiments that these

spotsrepresentedstepsin thepost-translational

modification process. The presence ofmultiple

glycoprotein spots in virus preparations may

indicate either that post-translational modifica-tionoccursinthe virusorthat the virus contains

forms of thesameglycoproteinsthatare

glyco-sylated to different extents. Our analysis also

indicated that certain bandsonone-dimensional

SDS-PAGE gelsmay represent more than one

polypeptide.Twoor morespots were present at

the positions occupied by bands VP12, VP13,

VP18, VP19 and VP22, and the results of a

simple SDS-PAGE analysis of these proteins

mustbeinterpretedwith caution.

We found that IBRV virions were resolved

into two distinct bands on potassium tartrate

gradients (Fig. 2A). An SDS-PAGE analysis of

the proteins indicated that the lower,

DNA-bearing virions containedproteinsVP17, VP22,

and VP24 which were absent from the empty

virions. Bothcomponents were probably

enve-loped since the viral glycoproteinswere

associ-ated with both of them. Asimilarphenomenon

has been observed with herpes simplex virus. Thus, nucleocapsids of herpes simplex virus band as two separate components on sucrose

gradients. The faster-sedimenting component

comprisesparticlesthat containDNA, whereas the more slowly sedimenting peak consists of

emptycapsids. In additiontocontainingDNA,

the fullparticlesalso containafew

DNA-asso-ciatedpolypeptidesthatare notpresentin the

emptyparticles(10).

Ouranalysisofviral proteinsininfected cells

wasmadesimplerby the drastic decreaseinthe

rate of host cell protein synthesis caused by

IBRV infection, such that radioactive precursors

added to the infected cells 6 h after infection

wereincorporated almost exclusivelyinto

virus-induced polypeptides. In addition to the 25

structuralpolypeptides, infectedcellscontained

atleast15nonstructuralpolypeptides.Although

atleastsomeoftheseareprobably

virus-speci-fied enzymes, others may represent precursors

of other structural and nonstructural

polypep-tides.

Herpesviruses, whichpossess the capacity to

code for more than 100 genes, have aparticularly

complex temporal pattern of gene expression.

Thus, herpesvirus-specified genes, transcripts,

PROTEINS SPECIFIED BY IBRV 377

and polypeptides can be classified as a or

im-mediateearly,,B orearly, and-y orlate,

depend-ing upon the order of expression during the

infectious cycle.InthecaseofmostDNAviruses

andatleastsomeherpesviruses, suchashuman

cytomegalovirus (30), murine cytomegalovirus

(16; J. K. Chantler, personal communication),

and pseudorabies virus (3), the synthesis ofy

polypeptides also requires the prioronsetof viral

DNAsynthesisintheinfectedcells.In contrast

tohumancytomegalovirus, murine

cytomegalo-virus, andpseudorabies virus, inhibitors of DNA

synthesis donotpreventthesynthesisofy

poly-peptides and theassemblyofempty

nucleocap-sids in herpes simplex virus-infected cells (23).

In this respect IBRV more closely resembles

humancytomegalovirus, murine

cytomegalovi-rus,and pseudorabies virus. Atleast five

poly-peptides (GVP1, GVP3, NSe, VP8, and VP13)

eitherwere missingfromara-C-treatedcellsor

weremade indrasticallyreducedamounts.

Sim-ilar resultswereobtained when viral DNA

syn-thesis was inhibited in infected cells by

phos-phonoformic acid or bromovinyldeoxyuridine

(datanotshown).

Wealso identified threeapolypeptides (NSb,

NSc, and NSg), whichweresynthesized inlarge

amountsaftercycloheximidewasremoved from

cultures of infected cells.Although NSg has the

sameelectrophoretic mobilityonSDS-PAGEas

VP8,webelieve that it isadifferentpolypeptide

sinceVP8wasidentifiedasaypolypeptideand

was probably not synthesized until after the

onsetof viralDNAsynthesis.

The glycoproteins specified byherpesviruses

are ofparticular interest since they are social

proteins that are involved in interactions

be-tween virions and host cells, between infected

cells and the immunesystem, and between

in-fectedcells. Theherpessimplexvirus

glycopro-teins gC, gD,gA, andgB (allyclass) have been

shown to be involved in immune recognition,

andantibodies directedagainst these

glycopro-teinscanmediate immunocytolysisin

conjunc-tion withcomplementonmononucleareffector

cells (16). None of theIBRV glycoproteins

be-longed to the a class, but surprisingly at least

oneIBRVglycoprotein (GVP11)behaved likea

,Bpolypeptide since itwassynthesized (labeled

with [3H]glucosamine) in the absence of viral

DNAsynthesis. We are now studyingthe roles

played bythe various IBRVglycoproteins, and

in particular GVP11, in immune recognition.

TheabilityofGVP11 toparticipatein immune

cytolyticprocesses would havespecial

implica-tions in interacimplica-tions with non-productivity or

latently infectedcellsin which viral expression

ispresumablyrestricted to the a and,Bclasses.

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378 MISRA, BLUMENTHAL, AND BABIUK

ACKNOWLEDGMENTS

We thank Kris Komendant for her excellent technical assistance.

Funds from the Medical Research Council of Canada made thisproject possible.

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