Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells.

(1)

0022-538X/88/114239-10$02.00/0

Expression of Bovine Herpesvirus

1

Glycoproteins

gI

and

glll

in

Transfected Murine Cells

DAVID R. FITZPATRICK,' TIM ZAMB,2 MICHAELD. PARKER,'3 SYLVIAVAN DRUNEN LITTEL-VAN DEN

HURK,"13 LORNE A. BABIUK,13* AND MICHAEL J. P. LAWMAN'13

Department of Veterinary Microbiology, University of Saskatchewan,' and the Veterinary InfectiousDisease

Organization,3* Saskatoon, Saskatchewan, Canada, S7NOWO, andDepartment ofVeterinary Sciences,

Universityof Nebraska, Lincoln, Nebraska 685832

Received9May1988/Accepted 19July 1988

Genesencoding twoof the major glycoproteinsof bovineherpesvirus 1 (BHV-1), gIand gIll, werecloned intothe eucaryotic expressionvectorspRSVcatandpSV2neoand transfectedinto murine LMTK-cells,and cloned cell lines were established. The relative amounts ofgI or gIII expressed from the two vectors were

similar.ExpressionofgIwascellassociatedandlocalizedpredominantlyintheperinuclear region,but nuclear andplasmamembranestainingwasalso observed. ExpressionofgIwasadditionallyassociated withcell fusion and the formation ofpolykaryons and giant cells.ExpressionofgIIIwaslocalizedpredominantlyinthenuclear and plasmamembranes. Radioimmunoprecipitation inthepresence orabsence oftunicamycin revealed that

the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights

similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both

recombinantglycoproteins were glycosylatedtoalesserextentthanwerethe forms found in BHV-1-infected

bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was

identified; for glll,adeficiencyIn0-linkedglycosylationwasimplicated.Thereactivity patternofapanelof gI-andgIII-specific monoclonal antibodies, including six which recognizeconformation-dependent epitopes,

wasfoundtobe unaffectedbytheglycosylationdifferences andwasidentical for transfectedorBHV-l-infected

murine cells. Useof thetransfected cellsastargetsinimmune-mediatedcytotoxicity assaysdemonstrated the

functional recognition of recombinant gI and glll by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus

verifyingtheantigenic authenticity of the recombinant glycoproteinsand theimportant roleofgIandgIll as

targets of the immuneresponsetoBHV-1 in this murine modelsystem.

Bovineherpesvirus 1(BHV-1)specifies fourmajor glyco-proteins,tentatively designatedgI, gIl,

glll,

and gIV, which

are homologous to the herpes simplex virus (HSV)

glyco-proteinsgB, gE, gC, and gD, respectively (50; T. Zamb et

al., manuscript in preparation). Ofthese glycoproteins, gI,

gIll,and gIVhave beenidentified asthemajor immunogens

recognized by sera from cattle infected with BHV-1 (49).

Furthermore, immunization with anyofthese three

glyco-proteins, individually orincombination, hasbeen shown to

induce significant protection against BHV-1 infection in

cattle (3). However, little is known ofthe cellular immune responses toBHV-1glycoproteins, whicharelikelytobe the

mostimportantresponsesmediatingtheobserved protection

(3, 42). In particular, the induction ofmajor

histocompati-bilitycomplexantigen-restricted Tlymphocytes by the

gly-coproteinimmunogenswould be aprerequisite forallofthe

acquired cell-mediated immune defense mechanisms (42).

A second area which is poorly understood concerns the

biological function(s) of the glycoproteins of BHV-1.

Al-though the homology ofgI, gII, gIII, and gIVto the HSV

glycoproteins notedabove is well

cstablished

at the

nucleo-tide and amino acid sequence levels (Zambetal., in

prepa-ration), it has not yet beendirectly demonstrated that the

glycoproteinsof BHV-1 possess homologousfunctions. The

HSVglycoprotein-associated activities of virus attachment (15), virus penetration (16, 43), cell fusion (31, 36), virus

assembly (1), complement factor C3b binding (12), and

immunoglobulin G (IgG) Fc binding (23) are intimately

*Corresponding author.

associated with the pathogenesis of infection, and it is thereforeimportanttodetermine whether such functionsare

conserved in BHV-1.

To analyze the immunobiology ofthe major BHV-1

gly-coproteins in more detail, we have initiated studies to

produce each glycoproteinby recombinant DNA,

mamma-lian cell-based expression systems. The establishment of stable mammalian cell lines which constitutively express

authentic individual BHV-1glycoproteins wouldbe

particu-larlyuseful in studies todetermine thebiological function(s)

of each glycoprotein and to dissect the specificities of individual cellular immune defense mechanisms directed

against BHV-1.

In this report, we describe the derivation oftransfected, cloned murine cell lines expressing BHV-1 gI and gIll. Subcellular localization of expression was determined by

immunocytochemistry. Structural andantigenic

characteris-tics of the recombinant glycoproteins were examined by

comparison with the glycoproteins produced in

BHV-1-infected bovine cells, by analysis of the reactivities ofa

panel ofglycoprotein-specific monoclonal antibodies, and,

for gI, by testingfor the biologicalfunction of cell fusion.

Use of the cell lines in

preliminary

analysisof thespecificity of

antibody-

andcell-mediated immune responsestoBHV-1 ina mousemodel systemis described.

MATERIALS ANDMETHODS

Reagentsand media. Restriction enzymes, T4 DNA poly-merase,T4DNAligase,calf intestinalalkalinephosphatase,

phosphorylated BglII linkeroligonucleotides,

deoxynucleo-4239

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

4240 FITZPATRICK ET AL.

A. pRSVgI

ATG TG

---. 4 t

E

B. pSV2gI

C. pRSVgIII

D. pSV2gIII

L7YZIZIZm

4

E

t E

t

TG

+ 4

B

ATG TAG + -* +

t

B I

ATG TAG

lo

t

B/Ba

zIzzzIz-E E

4

B

4

B/Ba

FIG. 1. Structure ofgI and gIll expression plas

tions. The origins of DNA sequences included in I

plasmids are represented as follows: Ffiz, pBR322

E, SV40; _, BHV-1; ED, TnS (20, 45). The codons ofgIandgIll areindicated (Zambetal.,inpr(

the directionof transcription from the RSV andSV40

arrowed (11, 55). Restriction endonuclease cleaN

EcoRI, B, BglII; B/Ba, BgllI-BamHI sites destroyec

side triphosphates, and protein A-Sepharosewe

from Pharmacia, Dorval, Quebec, Canada, recommended by the manufacturer. Other cl

reagents for DNA manipulations, transfections

analysis were purchased from Sigma Chemi

Louis, Mo., and used in the standard methods Maniatis et al. (30) and Davis et al. (9), excep

otherwise below. Cell culture media, fetal b (FBS), G418 and other cell culture reagents M

from GIBCO/BRL, Burlington, Ontario, Cana ies, wheat germagglutinin, avidin-biotin immui

staining kits, and other reagents for enzyme in

werepurchasedfromDimensionLaboratories,

Ontario, Canada, and used as recommended

facturer. Radioisotopically labeled compounds forfluorographywerepurchased from Amersh;

Ontario, Canada.

Plasmid constructions. The complete coding BHV-1gIwasexcisedfromasubcloneof pSDI

etal., inpreparation)andinserted intotheexpr

pRSVcat (20) in place of the cat gene by li,

3,300-base-pair (bp) BglII-BamHI gI gene fragi

HindIII-HpaI sites of pRSV cat after these si converted into a unique BgII cloning site

repair, BglII linker addition, and BglII dige manipulations removed thenormalviral promc of the gI gene and placed the start codon of

approximately 100 bp downstream of the R virus (RSV) promoterandapproximately 70

bp

of thetranscriptional startsiteassociated with (55;Fig.1A). Approximately 480bplaybetwe codon and thesimianvirus 40(SV40)-based

pol

signals remaining inthe expression vectorafte of the cat gene. A polyadenylation signal of]

approximately30bpdownstreamof thegIstop etal., in preparation) was retained inthis cons

gI gene was similarly subcloned into the expr

pSV2neo (45) in place of the neo gene by li

3,300-bp BglII-BamHI gI gene fragment into

SmaI sites ofpSV2neo after these sites had be into a unique BglII cloning site as described

3A These manipulations placed the gI gene start codon approx-imately 130 bpdownstream of theSV40early promoter and t

t

approximately

100

bp

downstream of the

transcriptional

B/Ba E start site associated with this promoter (11; Fib. 1B). Fol->A lowing the

gI

genestopcodon wereapproximately 430bpof ________lnoncoding BHV-1 DNA, 170 bp ofnoncoding TnS DNA, the B/Ba + sequencesencoding the SV40 smallt-antigenintron,and the B/Ba E SV40polyadenylation signals (45).

Thecompletecoding sequence of BHV-1gIllwasexcised

Ikb from a subclone of pSD113 as a 2,400-bp

BamHI-EcoRI

t

fragment

(32;

Zamb et

al.,

in

preparation),

treated with T4 E DNA

polymerase, ligated

to

BglII linkers, digested

with BglII, and then cloned into pRSVcat and pSV2neo as

______>

described for BHV-1 gI

(Fig. 1C

and

D).

In the

pRSVgIII

construction, thegIll gene start codon wasplaced approxi-E mately 140 bp downstream of the RSV promoter and 110 bp smid construc- downstream of the transcriptional start site associated with the expression this promoter. Approximately 850 bp of DNA lay between 2; rmr, RSV; the

glll

stop codon and the vector-associated

polyadenyla-startandstop tion signals. In the pSV2gIII construction, the gIII start

eparation), and codon waspositioned approximately 170 bp downstream of

promoters are the SV40 early promoter and 140 bp downstream of the

iage

sites: E, transcriptional startsite. Followingthe

glll

stopcodonwas I by ligation, approximately 800 bp of BHV-1 DNA, plus the

Tn5

and

SV40 sequences noted above forpSV2gI.

~re

purchased Plasmid DNA was prepared for transfection by equilib-and used as rium banding in CsCl-ethidium bromide gradients and

ster-hemicals and ilized by ethanol precipitation.

,,

and protein Cells and virus. Madin-Darby bovinekidney (MDBK)and ical Co., St. murine 3T3 cells were cultured in Eagle minimal essential described by medium supplemented with 10%FBS. Murine LMTK-and

t when noted L929 cells andcultured inDulbecco modifiedEaglemedium )ovine serum supplemented with 5% FBS. Virus stocks of BHV-1 P8-2 vere obtained were grown in MDBK or Georgia bovine kidney cells as

ida. Antibod- previously described(4). Virus stocksof vaccinia virus WR noperoxidase were grown in BS-C-1 cells as previously described (29). nmunoassays Transfections. LMTK-cellsweretransfected with

expres-Mississauga, sionplasmid constructionsbyamodified calciumphosphate by the manu- precipitation procedure. LMTK- cells at approximately andreagents 50% confluency were rinsed and incubated at 37°C in fresh am, Oakville, growth medium for 3 h before transfection. Calcium phos-phate precipitates of plasmid DNAwere prepared as

previ-sequence of ously described (22, 54), with pSV2neo DNA incorporated L06 (32; Zamb into each precipitate as acotransfecting selectable marker. -essionvector Controlprecipitates were preparedwith pSV2neoor salmon gation of the sperm DNA only. Medium wasremovedfromthe cells, and ment into the the DNAprecipitateswere added andadsorbedfor 45 minat ites had been room temperature. Growth medium was then

added,

and by blunt-end adsorption continued at 37°C in a 4% CO2 atmosphere

(7).

-stion. These After 4 h the medium was removed, and the cells were )terupstream exposed to 20% glycerol shock (13) for 2 min at room

f the gI gene temperature and then incubated at 37°C in growth medium 'ous sarcoma supplemented with 8 mM sodium butyrate (19). After 16to

)downstream 24 h the supplemented medium was removed and

replaced

this promoter with growth mediumfor48 h. The cells were then

passaged

en thegI stop in selective growth medium containing 400 ,ug of G418 per lyadenylation ml, which wasreplaced every 3 to 5days.Resistantcolonies r the removal appeared in 10 to 14 days at afrequency of

approximately

BHV-1 origin

1i-'

by this method. The colonies derived from each trans-codon(Zamb fection were pooled and cloned by

limiting

dilution at least

truction. The once before screening.

ession vector Immunocytochemistry assay and ELISA. G418-resistant igation of the LMTK- cell clones were seeded ontoglass chamber slides the HindIII- (Miles Laboratories, Rexdale, Ontario, Canada) and96-well en converted plastic tissue culture plates (Nunclon, Roskilde,

Denmark),

for pRSVcat. which had been precoated with 2 ,ug ofpoly-L-lysine

hydro-

E--J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.612.61.296.72.230.2]

(3)

bomide per cm2, and grown to confluency. For

BHV-1-infected control cells, MDBK or LMTK- cells were

simi-larly seeded onto poly-L-lysine-coated slides and plates, grown to 80%confluency, and then infected with BHV-1 at

a multiplicity of infection of 1. After 1 h of adsorption at

37°C, fresh medium containing 2% FBS was added, and

incubationwascontinuedforafurther12 to 18 h,forMDBK

cells, or for a few minutes, for infected LMTK- cells.

Transfected LMTK- cell clones and control cells were

either fixed and permeabilizedwithmethanolat-20°C for15

min and then washed in Hanks balanced salt solution

(HBSS)or,for surface expression studies,washed inHBSS

without being fixed. Nonspecificbinding sites were blocked

by adding heat-inactivatednormalequineserumdiluted1:75

in HBSS and incubating the mixture at room temperaturefor 1 h. The blocking solution was removed, and biotinylated wheat germ agglutinin ormonoclonalantibodies specific for gIandglll (52) werediluted 1:1,000 inHBSS and added to

theslidesand plates,whichwereincubatedat room

temper-aturefor1 h. The slides and plates were then processed with

an avidin-biotin-enhanced immunoperoxidaseassay kit

spe-cific for mouse IgG (Vector Laboratories, Burlingame,

Calif.)asrecommendedby the manufacturer,up to the final

substrate development step. For slides, the final substrate

was 50 mM Tris hydrochloride (pH 7.5)-0.01% H202-1.7

mM NiCl2-1 mg of 3,3'-diaminobenzidine

tetrahydrochlo-ride per ml. The substratereactionwas stopped after 5 min

ofincubationat roomtemperatureby rinsingtheslides in tap

water. For enzytne-linked immunosorbent assays (ELISAs)

the final substrate was 0.1 M citric acid (pH 4.0)-0.015%

H202-1 mg ofABTS [2,2'-amino-di-(3-ethylbenzthiazoline

sulfonate)] (6) per ml. The ELISA substratereactionswere

stopped aftera5- to 10-minincubationatroom temperature

by addition of sodium dodecyl sulfate (SDS) to a final

concentration of5%, and theA405ofeachwellwasread in a

plate reader.

Radioimmunoprecipitation. To

radiolabel

cellularproteins,

clones of transfected LMTK- cells at approximately 80%

confluencywereincubatedat37°C for6h inmethionine-free

Dulbecco modified Eagle medium supplemented with 2%

FBS. Forglycosylation inhibition studies,

tunicamycin

was

included at this point at a final concentration of 2 ,ug/ml.

After 6 h ofincubation,

[35S]methionine

wasadded to a final

concentration of50 ,uCi/ml, and the cells were then

incu-bated for an additional 18 h. BHV-1-infected MDBK cells were radiolabeled by a similar method, as previously de-scribed (47).

Radiolabeled cells were harvested by scraping, washed

withHBSS, and suspended inmodifiedRIPAbuffer(50 mM

Tris hydrochloride [pH

8.0],

150 mM

NaCl,

1% sodium

deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM

phenyl-methylsulfonyl fluoride). Afterincubationonice for15min,

the cell suspensions were sonicatedand then

centrifuged

at

75,000 x g for 1 h at

4°C.

The supernatants werecollected,

gI-orgIll-specificmonoclonal

antibody

ascites fluid(49)was

added to a final dilution of1:20, SDS was added to a final

concehtration of0.2 to 0.5%, and the samples were

incu-bated for 16 to 18 h at 4°C on a rocking platform. Coated

protein A-Sepharosebeads wereprepared by swelling

lyoph-ilizedprotein A-Sepharosebeads inmodified RIPA buffer at

a concentration of 10 mg/ml for 1 h at 4°C on a rocking

platform, then adding rabbit IgG antimouse IgG to a final

concentrationof 800,ug/ml, andincubatingthe

mnixture

for a

further 16 to 18 h. After incubation, unbound rabbit IgG anti-mouse IgG was removed from the coated protein

A-Sepharose beads by three washes with modified RIPA

buffer. Approximately10 mgof coatedprotein A-Sepharose beads wasaddedtoeach mixture ofradiolabeled celllysate plus monoclonal antibody, and thesamples wereincubated

at4°Con arockingplatform.After 3to4h,thesampleswere

washedfourtimes withmodifiedRIPAbuffer, suspendedin

reducing sample buffer (62 mM Tris

hydrochloride [pH 6.8],

2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.01%

bro-mophenol blue), and boiled for 4 min. Samples were

sepa-rated by

electrophoresis

in SDS-10%

polyacrylamide gels

and

fluorographed

(50).

Antibody complement cytotoxicity. Transfected murine

clones wereseeded into96-wellround-bottom

plastic

tissue

culture plates at a density of 2 x 103 cells per well and

incubated for24 hat

37°C

in

growth

medium

containing

1.5

pRCi

ofNa251CrO4 per well. The plates were washed three

times,

and control, gI-specific, or

gIII-specific

monoclonal

antibodies were added at various dilutions in Dulbecco

modified Eagle medium

containing

2% FBS and 1 ,ug of

actinomycinD per ml. Thetransfectedcells,like allnormal

nucleated cells, are resistant to complement attack in the absenceof metabolicinhibitors suchas

actinomycin

D(6;M.

Campos

and D. R.

Fitzpatrick, unpublished

observations). After 2 h of incubation at

37°C, freshly

thawed rabbit

complement

(Cedar

Lane,

Hornby, Ontario,

Canada), at

various

dilutions,

wasadded.Controlwellsfor

calcjilation

of

totalreleasableradiolabelreceived 3%Triton X-100 instead

of

complement.

After 90 min ofincubationat

37°C,

50% of

the supernatant fluid from each well was harvested and

counted, and the

specific

release was calculated as

previ-ously described (14, 34).

Cytotoxic

T-ceHl cytotoxicity.

C3H/HeJ

(H-2k)

or Balb/c

(H-2d)

mice were immunized

intraperitoneally

with

approx-imately 108 PFU ofBHV-1 at 8and 11 weeks ofage. At 3

weeks after the second

immunization,

the spleens were

excised and cell

suspensions

were

prepared by gentle

ho-mogenization.

The

suspensions

were treated with 0.83%

ammonium chloride to remove

erythrocytes,

washed,

counted,

scored for

viability,

and seeded into 6-well tissue culture

plates

at aconcentration of

approximately

2 x 106

cells per well in RPMI 1640 medium

containing

10%

FBS,

25

mM

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

acid

(HEPES), and 5 x 10-5 M

2-mercaptoethanol.

The cells

wererestimulated with 2 x 106 PFUof BHV-1 per well and incubated at37°Cina humidified5% CO2 atmospherefor 6 days.

L929and3T3cellstobe usedastargetswere

suspended

in RPMI 1640 medium and infected with BHV-1 or vaccinia

virus at a multiplicity of infection of 5 for 1 h at

37°C.

Infected targets, uninfected controls, and transfected cells

were then labeled with

Na251CrO4

for 1 h at

37°C.

The labeled target cellswerewashed three times with RPMI 1640 medium

containing

5%

FBS,

25 mM

HEPES,

and 5 x 10-5

M

2-mercaptoethanol

andthenseeded into U-bottom

micro-dilution

plates

at 104cells per well.

Restimulatedeffectorcellswerewashed, counted, scored

for

viability,

and addedtothe

plates containing

radiolabeled targetsat various

effector-to-target-cell ratios,

with

quadru-plicate

wells used for each variable. The

plates

were incu-bated for7hat

37°C

in a5%

CO2 atmosphere,

supernatant fluidswereharvestedandcounted, andspecific

cytotoxicity

valueswere calculatedas

previously

described(27).

Immunizations with transfected cells and antibody titer

determinations. C3H/HeJ micewereimmunized

intraperito-neally with 1065 transfected cells

suspended

in 0.5 ml of

HBSS, without

adjuvant,

at 6, 10, and 14 weeks of age.

Pooled serawere obtained at 5,

8, 11,

and 15 weeks of age

on November 10, 2019 by guest

http://jvi.asm.org/

(4)

from groups of five identically immunized mice. BHV-1-specific antibody levels were measured by virus neutraliza-tion assay and ELISA as previously described (52).

RESULTS

Expression ofrecombinant gI andglllintransfected murine LMTK- cells. Approximately 120 limit-diluted clones from transfections of the fourexpression constructions described above, plus negativecontrol clones derived from a transfec-tion conducted with pSV2neo alone, were screened for expression ofBHV-1 gI orglll byELISA and an immuno-cytochemistry assay. The use of unfixed ormethanol-fixed and permeabilized cells in each assay revealed surface or surface plus intracellular glycoprotein expression, respec-tively.

ELISA was used to compare the relative amount of surface and intracellular gI or glll expression by clones derived from a single transfection and by clones derived fromtransfections with the different expression vectors. For 17 clones positive for gI expression and 35 clones positive for gIll expression, a similar range and distribution of ELISA readings was obtained with either pRSV- or pSV2-based constructions (data not shown).

Immunocytochemistryrevealed thatexpression ofgI was

localized predominantly intracellularly in a perinuclear re-gionwhich probablycorresponds to the Golgi apparatus and/ or roughendoplasmic reticulum of these cells, as evidenced bythe similarlocalization of wheat germ agglutinin (Fig. 2G and H). However, cell surface expression of gI was also visible (Fig. 2B), and some nuclear-membrane localization wasmanifest as faint ringsoutlining the nuclei (Figs. 2E and H),whichwere notdetectable in negative controls (Fig. 2D). In addition, clones expressing gI exhibited a high degree of cellfusion, polykaryon formation, nucleusfusion, and giant-cellformation (Fig. 2E, J, and K), which was notapparent in clones expressing gIl or negative control clones. Expres-sion ofgIll was localized predominantly in the nuclear and plasma membranes, although diffuse cytoplasmic staining was also evident (Fig. 2C, F, and I). The subcellular distri-butions of recombinant gI and gIll are similar to those observed for theseglycoproteins in BHV-1-infected bovine cells (37),although the perinuclearaccumulation ofgI in the transfected murine cells appears to be greater than that observed in infected bovine cells.

Comparison ofrecombinant gI and glll produced in trans-fected murine cells with gI and gIll produced in BHV-1-infected bovine cells. Radioimmunoprecipitation ofgl from BHV-1-infected bovine cells revealed three major protein bands of molecular weight (MW) approximately 130,000, 75,000, and 55,000 (Fig. 3, lane 2), which correspond, respectively, to the intact uncleaved glycoprotein and the two cleavagefragments which are linked by disulfide bond-ing in themature nondenatured molecule (50). Only the last two cleavagefragments were precipitated from two clonesof murine cells transfected with gI expression plasmids, indi-cating thatproteolytic cleavage ofgIoccurred tocompletion inthese cells (Fig. 3, lanes 3 and 4). Inaddition, the larger of the twofragments produced in the transfected murine cells was slightly lower in MW than the equivalent fragment produced in infected bovine cells. Identical results were obtained with a number of other clones positive for gI expression (data not shown).

Radioimmunoprecipitation of gIll from infected bovine

cells yielded two major bands of MWapproximately 99,000 and 73,000 (Fig. 4, lane 2). Thesecorrespond, respectively,

tothematureglycosylated gllland itspartially

glycosylated

precursorform(50). Onlytheformer bandwas

precipitated

from clones of murine cellstransfected withtheglll expres-sion

plasmids, suggesting

that the precursor

form(s)

of

glll

is

moreefficientlyprocessed to maturemoleculesin themurine

cells. As observed for gI, recombinant glll had a

slightly

lower MWthanthematureform ofglll produced ininfected

bovinecells

(Fig. 4,

lanes 3 and

4).

These results werealso

verified by analysisof a numberofotherclones positive for

gIll expression (data notshown).

Analysis of the proteins precipitated from cells treated

with an N-linked glycosylation inhibitor, tunicamycin, was

conducted to compare the N- and 0-linked

glycosylation

patternsof the recombinant andinfected-cell

glycoproteins.

Radioimmunoprecipitation with gI-specific antibodies

yielded a single band of MW approximately 105,000 from

both

infected

bovine cells and gI-transfected murine cell

clones, although

additional

partially

glycosylated

products

ofMW

approximately

45,000 to50,000 also accumulated in thetransfected cells(Fig. 3, lanes 7 to 9). Theslightlyhigher MW of the 105,000-MW band in SV2gI-transfected cells

(Fig.

3, lane 9) is an artifact ofsample volume differences and was not observed in other experiments. The

105,000-MW band corresponds to the nonglycosylated, uncleaved form ofgI,which accumulatesowingtothedependenceofgI proteolytic cleavage on N-linked glycosylation and/or

asso-ciated function(s) which are blocked by tunicamycin (47).

The identical MW of this band in both infected bovine cells

and transfected murine cells indicates that no 0-linked

oligosaccharides are added to gI in either cell type and

suggests that the MW differences described above for

un-treated cells may be due todifferences inN-linked

glycosyl-ation.

Radioimmunoprecipitation of gIll from

tunicamycin-treated, BHV-1-infected bovine cells yielded two bands of

MWapproximately 80,000 and57,000 (Fig. 4, lane 7). These

correspond to a glycosylated form of gIll, containing only

0-linked oligosaccharides, and its nonglycosylated

precur-sor(47). OnlyanMW70,000band wasprecipitated from the

tunicamycin-treated, gIlI-transfected murine cell clones,

suggesting that any precursor forms of gIll are rapidly

processed in these cells and that the amount of 0-linked

oligosaccharides added to gIll is lower than that added in

infected bovine cells (Fig. 4, lanes 8 and 9).

The antigenic structure of the recombinant gI and gIII produced in the murine cell clones was analyzed with apanel

ofgl- and gill-specific monoclonal antibodies, the majority

of which have been mapped to different epitopes on these glycoproteins (51).Relativeantibodyreactivity was assessed by ELISA and immunocytochemistry on both fixed and unfixed cells and, for selected monoclonal antibodies, by radioimmunoprecipitation and/or flow cytometry. The immunocytochemistry results for methanol-fixed and per-meabilized cells are representative of all theassays used and are shown in Table 1. The reactivity pattern of the entire monoclonal antibody panel was identical for the recombi-nant and viral formsof gI and gIll, including two gI-specific and four gIll-specific antibodies which do not recognize denatured forms of these glycoproteins (51, 52). These results suggest that the primary, secondary, and/or tertiary structures of the recombinant glycoproteins, in the vicinity of the epitopes recognized by this panel of monoclonal

antibodies, areindistinguishable from those of the

glycopro-teins produced in BHV-1-infected bovine cells.

Recognition of gI and gIII by antibody and cell-mediated cytotoxicimmunedefense mechanisms. The antibody comple-J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

(5)

EXPRESSION OF BHV-1 gI AND glll GENES IN MURINE CELLS

IW6.

-4~ ~ ~ ~ A

t ..

A~~

k;ks

t

t.

I

* 9¶ '

7.*

*

, . U ... :'f'~

G

H

a

I

~~~~~~~.K

i

*t

J

t

K

'i4IiII

FIG. 2. Immunocytochemistry of transfected murine cells expressing BHV-1 gl or gill. LMTK cellstransfected with pSV2neo (A, D, and G), pSV2gl (B, E, H, J, and K), or pSV2gIII (C, F, and 1). Identical results were obtained with cells transfected with pRSV-based constructions. Live unfixed cells (panels A, B, and C) or methanol-fixed and permeabilized cells (panels D to K) were treated with monoclonal antibodies specificfor gl (panels A, B, D, E, H, J, and K) orgill(panelsA, C, D, F, and1) or with biotinylated wheat germ agglutinin (panel G) and then subjected to an avidin-biotin-enhanced immunoperoxidase staining procedure as described in Materials and Methods.

VOL. 62, 1988 4243

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.101.533.75.650.2]

(6)

1 2 3

4

5

6

7

8

9

10 200'-

-200

116-97p.

_-97

'116

66-04_m_

[image:6.612.72.284.75.218.2]

43N

-43

FIG. 3. Immunoprecipitation of gl from BHV-1-infected bovine cells and transfected murine cells. Lanes: 1 to 5, lysates from untreated [35S]methionine-labeled cells;6to 10, lysatesfrom tuni-camycin-treated and [35S]methionine-labeled cells. Uninfected MDBK cells (lanes 1and6), BHV-1-infected MDBK cells (lanes 2 and7),apRSVgl-transfected, cloned LMTK- cell line (lanes 3 and

8),apSV2gI-transfected, cloned LMTK- cell line (lanes 4 and 9),

andapSV2neo-transfected, cloned LMTK- cell line (lanes 5 and 10) areshown.Radiolabeled cell lysateswereimmunoprecipitated with a gI-specific monoclonal antibody (1F8 [52]), separated on

SDS-polyacrylamide gels, and fluorographed.

mentcytotoxicity results shown in Table 2 indicate that gI

andglllareexpressedonthe surfaces oftransfectedmurine

cell clonesatalevel andinamannerwhich isrecognized by complement-fixinggI- orglll-specific monoclonal antibodies

and which thereby renders the cells susceptible to attack

complement. The lower levels of lysis of cells expressinggI

are due primarily to the higher spontaneous release of

radioactive label fromunstable fusing cells and polykaryons.

Table 3 shows the results obtained in cytotoxic T-cell

cytotoxicity assays with transfected murine cell clones

ex-pressing gI or gIll as targets. In experiment 1, splenic

1 2 3 4 5 6 7 8 9 10

200'

116-

A_

97-_CNW

;_"

-200

4116

.197

TABLE 1. ReactivityofBHV-1-specific monoclonalantibodies withBHV-1-infectedortransfected murine cells

Monoclonal Reactivity Reactivitywith followingmurinecellsb:

antibody_antibody _denaturedwt

(epitope)" protein"i LMTK--BHV LMTK--gI LMTK-gIII

lBlO(g1-I) ++ + +

-3F3(gl-ll) ++ ++ ++

-lE11 (gI-III) ++ ++ ++

-1F8(gI-IVa) ++ ++ ++

-5G2(gI-IVb) ++ ++ ++

-5G11 (gI-IVc) ++ ++ ++

-lF1O (gI-V) + + + + + +

-2C5(gI-V) - + +

-1B4(gI-?) + + + +

-1F9(gI-?) - + +

-3H7(glll-Ia) ++ ++ - ++

lCll (glIl-Ib) ++ ++ - ++

3E3 (glll-Il) ++ ++ - ++

3F12(glll-Ill) ++ ++ - ++

1E2(gllI-IV) ++ + - +

3G8 (g1II-V) - + + - + +

1D6(gIII-VI) - + + - + +

2A11(glIl-VII) - ++ - ++

lF1l (glll-VIII) - ++ - ++

'Dataderived from references51 and 52. Fordefinition ofsymbols. see

footnoteb.

bReactivityasmeasuredbyimmunocytochemistry of methanol-fixedand

permeabilized cells. Identical resultswereobtained forcellstransfectedwith

pRSV-orpSV2-basedexpression plasmids. Reactivityscore: ++,positive, +,weaklypositive;-,negative.

lymphocytes from mice immunized and restimulated with

BHV-1 recognized andlysed histocompatible cells infected

with BHV-1. A portion of this activity was nonspecific

naturalkiller cell-like cytotoxicity,asevidencedbythelysis

of vaccinia virus-infected targets and

nonhistocompatible

targets; however, the marked restriction of cytotoxicity which occurred when nonhistocompatible target cellswere

usedprovided proof oftheinvolvement ofcytotoxic, major

histocompatibility complex-restricted T lymphocytes. The

results of experiment 2 confirm the above findings and establish the optimum effector-to-target-cell ratio for mea-surement of specific

cytotoxicity

as 50:1. Experiment 3 demonstrates thatrecombinant gIandglllexpressedbythe transfectedcelllines arerecognized bya

significant

propor-tion ofBHV-1-specific cytotoxic T

lymphocytes.

Note that

thelysis ofthenegativecontrol vaccinia virus-infected L929

so

66-43'

-66

.43

FIG. 4. Immunoprecipitation of glll from BHV-1-infected

bo-vinecells andtransfected murine cells. Lanes: 1to5, lysates from untreated [35S]methionine-labeled cells; lysates from

tunicamycin-treatedand[35S]methionine-labeled cells. Uninfected MDBK cells

(lanes 1 and 6), BHV-1-infected MDBK cells (lanes 2 and 7), a

pRSVgIII-transfected, cloned LMTK- cell line (lanes 3 and 8),a

pSV2glII-transfected, clonedLMTK-cellline(lanes4and9),and

apSV2neo-transfected,cloned LMTK- cell line(lanes5and10)are

shown. Immunoprecipitations were conducted as described in the

legend to Fig. 3, except that agIll-specific monoclonal antibody

[image:6.612.313.552.96.316.2]

(1D6 [52])was used.

TABLE 2. Antibodycomplement cytotoxicityof transfected murine cell clonesexpressing gI orglll

%Specific releasea

Negative

gI-specific

gilI

Targetcells control monoclonal specific

monoclonal antibody monoclonal

antibody' (lE11) antibody

_(11D6)

LMTK--pSV2neo 0.8 0.0 0.0

LMTK--pRSVgl 0.6 8.3 -C

LMTK--pSV2gl 2.3 25.0

-LMTK--pRSVgIlI 0.3 - 51.7

LMTK--pSV2gIIl 0.6 - 47.6

"Spontaneousrelease in thepresenceofcomplementalonedidnotexceed

17%of the totalreleasableradiolabel.

bBHV-1

glV-specific

monoclonalantibody3D9(52).

-,Notdone.

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.612.71.286.461.624.2] [image:6.612.317.556.583.694.2]

(7)

[image:7.612.58.554.86.298.2]

TABLE 3. Lysis oftransfected murine cells expressing gI or glll by BHV-1 specific cytotoxic T lymphocytes

%Specific release" with following effectorcell'andeffector-to-target-cellratio:

Expt Targetcells C3H/HeJ

BALB/c(50:1)

100:1 50:1 25:1 5:1 1:1

1 L929 -' 5.4 - - - 6.1

L929-BHV - 62.4 - - - 11.6

L929-vaccinia virus - 9.6 - - -

-3T3 - 3.4 - - - 3.4

3T3-BHV - 18.4 - - - 45.6

2 L929-vaccinia virus 12.3 8.8 6.4 2.1 2.4

-L929-BHV 72.0 63.6 34.3 21.0 14.2

-3T3-BHV 20.3 14.3 9.1 10.2 0.8

-3 LMTK--pSV2neo - 1.1 - - -

-LMTK--pRSVgI - 25.2 - - -

-LMTK--pSV2gI - 0 - - -

-LMTK--pRSVgIII - 23.1 - - -

-LMTK--pSV2gIII - 14.0 - - -

-L929-BHV - 63.1 - - -

-L929-vaccinia virus - 18.0 - - -

-L929 - 0 - - -

-aSpontaneous release fromtargetsdidnotexceed25% of the total releasable radiolabel. bEffector spleen cells were restimulated in vitro withBHV-1for6days.

-, Not done.

cells inexperiment 3 isabnormally highcompared with the for these glycoproteins in LMTK- cells. The induction of

results of experiments 1 and 2. We have also verified the significantlevels ofvirus-neutralizingantibodysupportsthe

specific recognition of gI and glll by using recombinant reactivity and cytotoxicity data which indicate that the

vaccinia virus-infected targets which express these glyco- recombinantglycoproteins are antigenically authentic.

proteins(M. J. P. Lawmanetal.,manuscriptinpreparation).

The different levels oflysis for pRSV- versus pSV2-based DISCUSSION

transfected cells, particularly for the gI-expressing cells,

does not correlate with the comparable total expression of Inthisreportwe havedescribed thederivation of cloned

the recombinant glycoproteins as measured by radioimmu- murine celllinesexpressing two ofthe major glycoproteins

noprecipitation and ELISA and maytherefore reflect quan- of BHV-1: gIandglll. Twodifferenteucaryotic expression

titativeand/orqualitativedifferences in the amount of proc- vectors were used for each glycoprotein gene owingto the

essed antigen(s) which is produced by the different existence ofconflicting reports concerningthe relative

effi-transfected cell lines and recognized by the cytotoxic ef- ciency oftheSV40- and Rous sarcomavirus-based

enhanc-fector cells inthisassay. er-promoter units in murine cells (8, 19) and owing to our

Immunogenicity of transfected cells in mice. Histocompat- intention to express these glycoproteins in a number of

iblemice immunized with transfectedcells in theabsence of mammaliancelllines in which cellspecificfactors may effect

adjuvant produced detectable BHV-1-specific antibody after the rate of expression from different enhancer-promoter

only one immunization (Table 4). Both ELISA and virus- units (18). Although we did not quantitate the gene copy

neutralizing antibody levels were significantly boosted by number, transcription rate, or other factors which may

secondary but notby tertiary immunization. Theinduction contribute to expression, we found that the final rate of

ofcomparableantibody levelswith cellsexpressinggIorgIll expression of mature gI or gIll from the pRSV cat- and

underthe control of different enhancer-promoterunits cor- pSV2neo-based constructions was similarin LMTK- cells.

roborates thedataabove, which suggestthat the SV40 and This observation was consistent for a number of clones

RSVelementsarequantitativelyequivalentexpression units derived from several transfectionswith eitherglycoprotein.

TABLE 4. Serologicresponsesof mice immunizedwithtransfected LMTK-cellsexpressingglorgilll

ELISAantibodylevelbatfollowing day Virus-neutralizing antibodylevel' atfollowing day

Cell after primary immunization: after primary immunization:

0 14 40 65 0 14 40 65

LMTK--pSV2neo <10 <10 80 160 <8 <8 <8 <8

LMTK--pRSVgI <10 1,280 20,480 20,480 <8 <8 32 64

LMTK--pSV2gI <10 1,280 20,480 20,480 <8 <8 32 64

LMTK--pRSVgIII <10 160 5,120 10,240 <8 <8 8 16

LMTK--pSV2gIII <10 80 5,120 5,120 <8 <8 8 8

aMicewereimmunizedintraperitoneallywith

106.5

cells ondays 0,28,and 56.

b ELISAtiters versus 0.5 ,ug ofpurified BHV-1per well.

c Virus-neutralizing antibodytiters versus 50PFUof BHV-1 inthe

absence

ofcomplement.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.612.54.552.613.700.2]

(8)

Theresults ofstudies analyzingtheintracellular distribu-tion, the biochemical and antigenic structure,and, forgI, a

functional property, indicate that therecombinant

glycopro-teins produced by the transfected murine cells are largely

authentic andare similartogI and gIII producedin

BHV-1-infected bovine cells, except for the following significant

differences. RecombinantgI was localized predominantlyin

aperinuclear region,whichprobablyincludesor liescloseto

the rough endoplasmic reticulum and/orGolgi apparatus of

LMTK- cells (Fig. 2G and H). Although perinuclear

accu-mulation of gI has also been described for BHV-1-infected

bovinecells (37), theaccumulation observedin the

transfec-tedmurinecell clonesexpressinggIappeared to be greater.

This observation is similar to thosereported forexpression

ofrecombinantHSVgBin Chinese hamsterovary and COS

celllines, in whichcell-associatedorperinuclear localization

was a prominent feature of the intracellular distribution of

this closely related protein (2, 38).

Three general explanations for the increased perinuclear

accumulation of gI in transfected murineLMTK-cellclones

arepossible. First, thegenes encoding gIin the transfected

murinecells may bestructurally flawedsuch that atopogenic

signal(s)inthe protein product, which isrequired for

trans-portofgI tothe nuclear and plasmamembranes,is absent or

notrecognized bythecellular transport machineryinalarge

proportion ofthemolecules produced. However, a

substan-tialamountofrecombinant gI issuccessfully transported to

the nuclearand plasmamembranes (Fig. 2), which

necessi-tates hypothesizing the presence oftwo or more copies of thegIgene, at least one of which is normal and oneof which is not. Given that the perinuclear accumulation gI was a

consistentfinding withall thepositive clones derived froma

number oftransfections with two different expression

plas-midconstructions, such a possibility isremote.

Asecond possible explanationis that LMTK- and MDBK

cells areinherently differentintranscriptional, translational,

and/or posttranslational processes involvedin thesynthesis

and transport ofgI. Hypothetically, this difference would

result, for LMTK- cells, in the production ofgI molecules

possessing hiddenorabsenttopogenicsignal(s) and/orin the

trapping of structurally authentic gI in the perinuclear

re-gion. The most likely difference, in this case, would be

posttranslational modification processes such as

glycosyl-ation, folding, and/or disulfide bondformation, which have

been shown to differ between mammalian cell lines and

which may affect protein transport (26, 30, 35, 44). This

explanation is plausible in light ofthe different pattern of

glycosylation ofgI detected in murine and infected bovine

cells(see below). However, thereproducibility ofincreased

perinuclear accumulation of gI or HSV gB in

LMTK-,

Chinese hamsterovary, and COS cells would argueagainsta

cell-specific mechanism, as does the fact that numerous

otherrecombinantglycoproteinshave been synthesizedand

transported correctly in LMTK- cells(5, 25,35, 39,46,53),

including BHV-1 glll, asdescribed above. In addition, the

treatmentofBHV-1-infectedbovine cells with the N-linked

glycosylation inhibitor tunicamycin was found to affect the

surface localization of

gI

rather than theintracellular

distri-bution (37).

The third possible general explanation is that efficient subcellular localization of gI requires a viral and/or

virus-influenced proteinor function which is absent or expressed

at low levels in cells transfected with the isolated

gI

gene.

This alternative could include a structural protein which

complexes with gI and controls its transport, as recently

reported for ahepadnavirus glycoprotein (39), or,possibly,a

viralorvirus-induced protein whichmayindirectly affect gI transport, such as

ICP4,

which has been reported to influ-ence the nuclear localization of some

delayed early

HSV

proteins (24). Conversely,thisalternativemayalsoincludea

virus-inhibited cellular function, for example, the

recently

characterized "molecular

chaperone"

proteins

(10,

41),

which maynormally retaingI in the perinuclear

region.

We regard the last twogeneral explanations as the most

likelyandarecurrently conductinganumberof

experiments

to testwhether'correctgIlocalizationoccursindifferent cell types and whether the localization in transfected LMTK-cells canbe altered

by

virus-associated functions.

Examination of the

glycoproteins

precipitated from

un-treated or tunicamycin-treated transfected murine cell clones andBHV-1-infectedbovine cells revealed that

recom-binantgI andgIll appeared tobeprocessedmore

efficiently

in the transfected murine cell clones than in the infected bovine cells. This was suggested by the lack of detectable precursorforms for bothrecombinant glycoproteins and the

complete proteolytic cleavage ofgI. Both recombinant

gly-coproteins

alsoappearedtobeslightlylessglycosylated than their counterpartsfrom BHV-1-infectedbovine cells. ForgI,

thisglycosylation differencewaslocalized to the MW75,000

fragment of the mature molecule by analysis of reduced

samples and to a difference in N-linked glycosylation

by

analysis of tunicamycin-treated cells (Fig. 3). For glll, a

deficit in 0-linked glycosylation was similarly identified

(Fig.4

). However, other subtle orcompensatory

processing

and modification differences were not completely ruled out

in eithercase.

Similarhypotheses, aspresented above for the

intracellu-lardistributionofgI, may bepostulatedto accountfor these

processingdifferences. In this instance, we initially favored

the possibility that cell-specific glycosylation,

oligosaccha-ride processing, and/or transport differences exist between

murine LMTK- cells and bovine MDBK cells, such as that

reported between murine L cells and avian cells (44) or

between mutated and normal L cells (46). In particular, such

differences would account for the reduced glycosylation of

both gI and gIII synthesized in murine cells. However, to account for the complete cleavage of recombinant gI, we

postulate that the lack ofaviral or virus-influenced protein

and/or function also affects the structure of this protein in the transfected murine cell clones. In support of this view, we

have observed a similar structure for gI expressed in a

variety of cell lines infected with a recombinant vaccinia

virus (S. van Drunen Littel-van den Hurk et al., manuscript in preparation). We hypothesize that in transfected or vac-cinia virus-infected cells, in which cellular protein synthesis is not as potently inhibited as in BHV-1-infected cells, the host cell proteolytic cleavage systems and, possibly, other posttranslational modification systems function more

effi-ciently to produce mature recombinant glycoprotein

mole-cules with more uniform structures. The experiments

out-lined above to investigate the factors influencing the subcellular localization of recombinant gI and gIII would also help determine the relative contributions of the postu-lated cell-specific and viral inhibition mechanisms to the synthesis and structures of these glycoproteins.

Despite the structural differences detected in both recom-binant gI and gIII, several qualities of these glycoproteins remained indistinguishable from those of gI and gIII synthe-sized in BHV-1-infected bovine cells. Significantly, these included functional and antigenic qualities, which are of prime importance for analyses of the immunobiological characteristics of the glycoproteins of BHV-1.

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

(9)

Recombinant gI exhibited the functional property of in-ducing cell fusion, polykaryon formation, nucleus fusion, andgiant-cell formation (Fig. 2). This propertyis knownto be associated with HSV gB (2, 31) but had not previously been definitively demonstrated for BHV-1 gI. In this

con-text,it is notable that the fusion activity for BHV-1 isnotas

greatasthat of HSV during normal infection of permissive

cells with these viruses. We have thereforeagain considered thepossibilities that cell-specific differences and/or the lack of viral or virus-induced functions are involved in the

expression of this activity in transfected murine cells. The planned experiments mentioned above mayalso yield infor-mation relevanttothis phenomenon. However, if the func-tional conservation between BHV-1gIand HSVgB demon-strated here is consistent for other viral proteins, several otherglycoproteins mayalso be involved in cell fusion and

polykaryon formation, for example, homologs ofHSV gD andgH (17, 36).

Theintegrity of the antigenicstructureof recombinantgI andglllwas probed by using a panel ofgI- orgIll-specific monoclonal antibodies in a number of binding assays, by using selected gI- and glll-specific monoclonal antibodies in antibody complement cytotoxicity assays, and by using murine BHV-1-specific T lymphocytes in cytotoxic T-cell cytotoxicity assays. The results of all thesetests indicated thatthe recombinant forms ofgIandglllwereauthentic and antigenically indistinguishable from the forms found in

BHV-1-infected bovine cells. This result is ofgreat

signifi-cance, since it indicates that the structural differences

de-tected in the recombinantglycoproteinsareimmunologically

irrelevant intermsof the criteria tested above, which include

a number of in vitro correlates of immune defense

mecha-nisms that may be activeagainst BHV-1 infection in vivo. The demonstration thatcytotoxic T lymphocytes induced by BHV-1maybe directedagainst cells expressinggIorglll

adds considerably to the relative target index of these proteins, whichwasalready thoughttobehigh owingtotheir early (gI)orhigh levels of(glll)expressiononthesurfacesof

infected cells(33, 48) and their relative immunodominancein

theantibodyresponse to BHV-1 (49). The significant levels

ofgI- and gIII-specific cytotoxic T-cell activity (Table 3),

whichwere elicited by immunization and restimulation with whole BHV-1 virus containingatleast 30 different structural proteins (33), suggestthat these glycoproteins may be rela-tively immunodominant for cytotoxic T-cell responses as

well as antibody responses. However, this result requires

confirmation in other strains of miceand,moreimportantly, in homologous bovine cytotoxic T-lymphocyte assay sys-tems.

Although the rational design of a subunit vaccine for

prevention of BHV-1 infections requires extensive analysis of the immunological qualities of many BHV-1 proteins, especiallynowthat internal and nonstructural viralproteins have been shown to be potential targets of cytotoxic

T-lymphocyte activity (21, 25, 40), the preliminary results obtained here withtwoof themajor glycoproteins of BHV-1 suggest that gI and glll are good potential candidates for

inclusion in such a vaccine. The availability of authentic

recombinant sources of these glycoproteins adds to this

potentialand should facilitate the furtherassessmentof their

suitability. A preliminary stepinthis directionwasmadeby

analyzing the immunogenicity of the transfected cells

ex-pressing gI orglll in histocompatible mice (Table 4). The

induction ofsignificant levels of BHV-1-specific and

virus-neutralizing antibody after only twoimmunizations ratifies

the above conclusions regarding the authenticity of these

recombinant glycoproteins and their candidature for

inclu-sion in asubunit vaccine againstBHV-1. We are currently

verifying

and

extending

these studies with other in vitro assaysand immunization experimentsin both mouse model

and bovine systems.

ACKNOWLEDGMENTS

We thank Andrew Potter, Gwen Hughes, Dirk Deregt, Manuel

Campos, and Jan van den Hurk for materials, assistance, and advice. The valuable contribution of Irene Kosokowsky in typing

themanuscriptis alsoappreciated.

This work wassupported bygrantsfrom the Medical Research Council and the National Science andEngineeringResearch Council of Canada. D.R.F. is supported by a Canadian Commonwealth

ScholarshipandFellowshipPlan Award. LITERATURE CITED

1. Addison, C., F. J. Rixon, J. W. Palfreyman, M. O'Hara, and V.G. Preston.1984. Characterization ofaherpessimplextype 1mutantwhichhasatemperaturesensitive defectinpenetration

of cells andassemblyofcapsids. Virology138:246-259. 2. Ali,M.A.,M.Butcher,and H. P.Ghosh. 1987.Expressionand

nuclearenvelopelocalization ofbiologically active fusion gly-coprotein gB ofherpes simplexvirus inmammalian cellsusing

clonedDNA. Proc.Natl. Acad. Sci. USA 84:5675-5679. 3. Babiuk,L.A.,J.L'Italien,S.vanDrunen Littel-van denHurk,

T.Zamb,M.J.P.Lawman,G.Hughes,andG. A.Gifford. 1987. Protection of cattle from bovine herpesvirus type 1 (BHV-1)

infectionby immunization with individual viralglycoproteins. Virology159:57-66.

4. Babiuk,L.A.,R.C.Wardley,and B. T. Rouse. 1975. Defense mechanisms againstbovine herpesvirus: relationshipof virus-hostcellevents tosusceptibilitytoantibody-complementlysis.

Infect.Immun. 12:958-963.

5. Blacklaws,B.A.,A. A.Nash,andG.Darby.1987.Specificityof theimmuneresponseof micetoherpessimplexvirus

glycopro-teinsB and Dconstitutively expressedonLcell lines. J. Gen. Virol.68:1103-1114.

6. Campbell,A.K.,and B. P.Morgan.1985. Monoclonal antibod-ies demonstrate protection of polymophonuclear leukocytes against complementattack. Nature(London) 317:164-166. 7. Chen, C., andH. Okayama. 1987. High-efficiency

transforma-tionof mammalian cells byplasmid DNA. Mol. Cell. Biol. 7: 2745-2757.

8. Choo, K. H., G. Filby, S. Greco,Y.-F. Lau,and Y. W. Kan. 1986. CosmidvectorsforhighefficiencyDNA-mediated

trans-formation and gene amplification in mammalian cells: studies with the humangrowthhormonegene.Gene 46:277-286. 9. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic

methods in molecular biology. Elsevier Science Publishing, Inc., NewYork.

10. Ellis, J. 1987. Proteins as molecular chaperones. Nature (London)328:378-379.

11. Fiers, W., R.Contreras, G. Haegeman, R. Rogiers, A.van de Voorde,H. vanHeuverswyn, J.vanHerreweghe, G.Volckaert, and M. Ysebaert. 1978.CompletenucleotidesequenceofSV40 DNA.Nature(London)273:113-120.

12. Friedman,H. M.,G. H. Cohen,R.J. Eisenberg,C. A.Siedel, and D. B. Clines.1984.GlycoproteinC ofherpes simplexvirus type 1acts as areceptorfor the C3bcomplementcomponenton

infected cells. Nature(London) 309:633-635.

13. Frost, E.,andJ.Williams.1978.Mappingtemperature-sensitive and host-range mutations of adenovirus type 5 by marker

rescue. Virology91:39-50.

14. Fujimiya, Y., B. T. Rouse, and L. A. Babiuk. 1978. Human

neutrophil-mediateddestruction ofantibody sensitized herpes

simplexvirus type 1 infected cells. Can. J. Microbiol. 24:182-186.

15. Fuller,A.O.,and P. G.Spear.1985.Specificitiesofmonoclonal and polyclonal antibodies that inhibit adsorption of herpes simplexvirus tocells and lackof inhibitionbypotent neutral-izingantibodies. J.Virol. 55:475-482.

on November 10, 2019 by guest

http://jvi.asm.org/

(10)

16. Fuller, A. O., and P. G. Spear. 1987. Anti-glycoprotein D antibodies thatpermit adsorptionbut blockinfectionbyherpes simplex virus 1 prevent virion-cell fusion at the cell surface. Proc. Natl. Sci. USA84:5454-5458.

17. Gompels, U., and A. Minson. 1986. Theproperties and sequence ofglycoprotein H of herpes simplexvirustype 1. Virology 153: 230-247.

18. Gorman, C. M. 1985. High efficiency gene transfer into mam-malian cells, p. 143-190. In D. M. Glover (ed.), DNA cloning: a practicalapproach, vol. 2. IRL Press, Oxford.

19. Gorman, C. M., and B. H. Howard. 1983. Expression of recombinant plasmids in mammalian cells is enhanced by so-diumbutyrate. NucleicAcids Res. 11:7631-7648.

20. Gorman, C. M., G. T. Merlino, M. C. Willingham, I. Pastan, andB. H.Howard. 1982. The Roussarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc. Natl. Acad. Sci. USA79:6777-6781.

21. Gotch, F., A. McMichael, G. Smith, and B. Moss. 1987. Identi-fication of viral molecules recognized by influenza-specific hu-mancytotoxic Tlymphocytes. J. Exp. Med. 165:408-416. 22. Graham, F. L., and A. J. van der Eb. 1973. A new technique for

theassay of infectivity of humanadenovirus 5DNA. Virology 52:456-467.

23. Johnson, D. C., and V. Feenstra. 1987. Identification of anovel herpes simplex virustype 1glycoproteinwhich complexes with gEand binds immunoglobulin. J. Virol.61:2208-2216. 24. Knipe, D. M., and J. L. Smith. 1986. A mutant herpesvirus

protein leads to a block in nuclear localization of other viral proteins. Mol. Cell. Biol.6:2371-2381.

25. Koszinowski, U. H., M. J. Reddehase, G. M. Keil, and J. Schickedanz. 1987. Host immune response tocytomegalovirus: products oftransfected viral immediate-early genes are recog-nized by cloned cytotoxic T lymphocytes. J. Virol. 61:2054-2058.

26. Lang, K., F. X. Schmid, and G. Fischer. 1987. Catalysis of protein folding by prolyl isomerase. Nature (London) 329:268-270.

27. Lawman, M. J. P., R. J. Courtney, R. Eberle, P. A. Schaffer, M. K. O'Hara, and B. T. Rouse. 1980. Cell-mediated immunity toherpes simplex virus: specificity of cytotoxic Tcells. Infect. Immun. 30:451-461.

28. Machmer, C. E., R. Z. Florkiewicz, and J. K. Rose. 1985. A single N-linkedoligosaccharideateither of the two normalsites issufficient for transport ofvesicular stomatitis virus G protein tothe cell surface. Mol. Cell. Biol. 5:3074-3083.

29. Mackett, M., G. L. Smith, and B. Moss. 1985. The construction andcharacterization ofvaccinia virus recombinants expressing foreign genes, p. 191-211. In D. M. Glover (ed.), DNAcloning: apractical approach, vol. 1. IRL Press, Oxford.

30. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: alaboratorymanual. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

31. Manservigi, R., P. G. Spear, and A. Buchan. 1977. Cell fusion induced by herpes simplex virus is promoted and suppressedby different viral glycoproteins. Proc. Natl. Acad. Sci. USA 74: 3913-3917.

32. Mayfield, J. E., P. J. Good, H. J. van Oort, A. R. Campbell, and D. E. Reed. 1983. Cloning and cleavage site mapping of DNA from bovineherpesvirus 1 (Cooper strain). J. Virol. 47:259-264. 33. Misra, V., R. M.Blumenthal, and L. A. Babiuk. 1981. Proteins specified by bovineherpesvirus type 1(infectious bovine rhino-tracheitis virus). J. Virol. 40:367-378.

34. Misra, V., J. E.Gilchrist, G. Weinmaster, L. Qualtiere, S. van den Hurk, and L. A. Babiuk. 1982. Herpesvirus-induced "early" glycoprotein: characterization and possible role in immune cytolysis. J. Virol.43:1046-1054.

35. Miyazaki, J., E. Appella, and K. Ozato. 1986. Intracellular transport blockade caused by disruption of the disulfide bridge inthe third domain of majorhistocompatibility complex class I antigen. Proc. Natl. Acad. Sci. USA 83:757-761.

36. Noble, A. G., G. T.-Y. Lee, R. Sprague, M. L. Parish, and P. G. Spear. 1983. Anti-gD monoclonal antibodies inhibit cell fusion

induced by herpes simplex virus type 1. Virology 129:218-224. 37. Okazaki,K., H. Kawakura,M.Okada,E.Honda, T.Minetoma, andT.Kumagi.1987. Intracellularlocalizationand transportof three different bovine herpesvirus type 1glycoproteinsinvolved inneutralization. Arch. Virol. 92:17-26.

38. Pachl,C., R. L. Burke, L. L.Stuve,L.Sanchez-Pescador,G.van

Nest, F. Masiarz, and D.Dina. 1987. Expression of cell-associ-ated and secretedforms ofherpes simplexvirustype 1 glyco-protein gB in mammaliancells. J. Virol. 61:315-325.

39. Persing, D. H., H. E. Varmus, and D. Ganem. 1986.Inhibitionof secretion ofhepatitis Bsurface antigen byarelatedpresurface polypeptide. Science 234:1388-1390.

40. Puddington, L., M. J. Bevan, J. K. Rose, and L. Lefrancois. 1986. N protein is the predominant antigen recognized by vesicular stomatitisvirus-specific cytotoxic T cells. J. Virol. 60: 708-717.

41. Rothman, J. E. 1987. Protein sorting by selectiveretention in the endoplasmicreticulum and Golgi stack. Cell 50:521-522. 42. Rouse, B. T., and L. A. Babiuk. 1978.Mechanisms of recovery

from herpesvirus infections-a review. Can. J.Comp.Med. 42: 414-427.

43. Sarmiento, M., M. Haffey, and P. G. Spear. 1979. Membrane proteins specified by herpes simplex viruses. III. Role of glycoprotein VP7(B2) in virion infectivity. J. Virol. 29:1149-1158.

44. Sheares, B. T., and P. W. Robbins. 1986. Glycosylation of ovalbumin in a heterologous cell: analysis of oligosaccharide chains of the cloned glycoproteinin mouse L cells. Proc. Natl. Acad. Sci. USA 83:1993-1997.

45. Southern, P.J.,and P. Berg. 1982. Transformation of mamma-lian cells to antibiotic resistance with a bacterial gene under controlof theSV40 early region promoter. J. Mol. Appl. Genet. 1:327-341.

46. Tufaro, F.,M.D.Snider, and S. L. McKnight. 1987. Identifica-tion and characterizaIdentifica-tionof a mouse cell mutantdefective in the intracellular transport ofglycoproteins. J. Cell Biol. 105:647-657.

47. van Drunen Littel-van den Hurk, S., and L. A. Babiuk. 1985. Effect oftunicamycin andmonensin onbiosynthesis, transport and maturation of bovine herpesvirus type 1 glycoproteins. Virology 143:104-118.

48. van Drunen Littel-van den Hurk, S., and L. A. Babiuk. 1985. Antigenicand immunogenic characteristics of bovine herpesvi-rus type 1glycoproteins GVP 3/9 and GVP6/lla/16, purified by immunoadsorbent chromatography. Virology 144:204-215. 49. van Drunen Littel-van den Hurk, S., and L. A. Babiuk. 1986.

Polypeptide specificity of the antibody response after primary andrecurrent infection with bovine herpesvirus type 1. J. Clin. Microbiol. 23:274-282.

50. van Drunen Littel-van den Hurk, S., and L. A. Babiuk. 1986. Synthesis and processing of bovine herpesvirus type 1 glyco-proteins. J. Virol. 59:401-410.

51. van Drunen Littel-van den Hurk, S., J. V. van den Hurk, and L. A. Babiuk. 1985. Topographical analysis ofbovine herpesvi-rus type 1 glycoproteins: use of monoclonal antibodies to

identify and characterizefunctionalepitopes. Virology 144:216-227.

52. van Drunen Littel-van denHurk, S., J. V.van denHurk,J. E. Gilchrist, V. Misra, and L. A. Babiuk. 1984. Interactions of monoclonal antibodies and bovine herpesvirus type 1 (BHV-1) glycoproteins: characterization of their biochemical and immu-nological properties. Virology 135:466-479.

53. Whang, Y., M. Silberklang,A.Morgan,S. Munshi, A.B.Lenny, R. W. Ellis, and E. Kieff. 1987. Expression of theEpstein-Barr virus gp350/220 gene in rodent and primate cells. J. Virol. 61:

1796-1807.

54. Wigler, M., A. Pellicer, S.Silverstein,R.Axel, G.Urlaub, and L. Chasin. 1979. DNA-mediated transfer of the adenine phospho-ribosyltransferase locus into mammalian cells. Proc. Natl. Acad. Sci. USA 76:1373-1376.

55. Yamamoto, T., B. deCrombrugghe, and I. Pastan. 1980. Iden-tification of a functional promoter in thelong terminal repeat of Rous sarcoma virus. Cell 22:787-797.

J. VIROL.