A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells.

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Vol. 62, No. 5 JOURNAL OF VIROLOGY, May1988, p. 1486-1494

0022-538X/88/051486-09$02.00/0

Copyright X) 1988, American Society for Microbiology

A

Herpes

Simplex Virus

Mutant

in

Which Glycoprotein D

Sequences Are

Replaced by

P-Galactosidase

Sequences

Binds

to

but Is Unable To Penetrate into Cells

MICHAEL W. LIGASANDDAVID C. JOHNSON*

MolecularVirology and Immunology Program, Department of Pathology, McMasterUniversity, Hamilton, Ontario, Canada L8N 3Z5

Received4November1987/Accepted11January1988

Herpes simplex virus(HSV) glycoprotein gDisamajorcomponentof the virion envelope and is thoughtto

play animportant role in the initial stagesof viral infection and stimulates the production of high titers of neutralizing antibodies. We assumed that gD playsanessential role in virusreplication, andsotocomplement viruses with mutations in the gDgene weconstructedacellline, denotedVD60, which is capable ofexpressing high levels of gD after infection with HSV. A recombinant virus, designated

F-gDoi,

in which sequences

encoding gD andanonessentialglycoprotein, gI,werereplaced by Escherichia coli ,I-galactosidasesequences, was selected on the basis that it produced blue plaques on VD60 cell monolayers underagarose overlays containing 5-bromo-4-chloro-3-indolyl-I-D-galactopyranoside (X-Gal). F-gDII wasable toreplicate normally oncomplementing VD60 cells. However, F-gD,I wasunabletoformplaquesonnoncomplementing Vero cells. Virionslacking gDwereproduced in normalamountsby Vero cells infected with F-gDfI, and the virusparticles

weredistributed throughout the cytoplasm andonthe cellsurface, suggesting that gD isnotessential forHSV envelopment andegress.Virionslacking gDwereabletobindtocells, butwereunabletoinitiatesynthesis of viral earlypolypeptides. Plaque production of F-gDfI particles lacking gDwasenhancedby polyethyleneglycol

treatment,suggesting that gD is essential for penetration of HSV into cells. Other HSV glycoproteins have been implicated in theentryof virus into cells, and thus thisprocessappearstoinvolvemultiple interactionsatthe cell surface.

Mostenvelopedanimal virusesincorporatealimited

num-ber of virus-encoded glycoproteins into host cell and viral

membranes. These polypeptides areutilized in the

envelop-ment of viral nucleocapsids and subsequentlyin the

recog-nition of and penetration into host cells. Viruses such as

influenza virus and vesicular stomatitis virus utilizeasingle glycoprotein in bindingtoandentering into cells (3, 20,38, 39).With these viruses, endocytosis precedes fusion ofthe virion envelope with the endosome membrane. Viral fusion

activity is triggered by the low pH of endosomes (reviewed in reference 20).

Other enveloped viruses, including Sendai virus, human

immunodeficiency virus, and herpes simplex virus type 1

(HSV-1), apparently enter cells by fusion of the virion

envelope directly withtheplasma membrane (9, 25, 34,38). However, incontrast tothe smallerRNAvirusesmentioned above, HSV-1

specifies

atleast seven membrane glycopro-teins (1, 4, 18, 27, 31). Fourofthesepolypeptides, gC, gE, gG, and

gI,

have been found to be dispensable for virus

replication

in cultured cells (12, 18, 19, 23, 28, 37). Ofthe three remaining glycoproteins, gB, gD, and gH, there is

evidence from studies involving virus mutants that gB and

gHareessentialforvirus entry intocellsorvirusspread (10, 17, 29; P. J.Desai,P. A.Schaffer, and A. C. Minson, Abstr. 12thInt.HerpesvirusWorkshop, p. 111, 1987). Experiments

involving monoclonal antibodies and liposomes suggested that gD might act as a virus attachment component or in virus entry into cells (8, 9, 12a, 16, 24).However, numerous attempts to isolate viruses with mutations in the gD gene

*Correspondingauthor.

have been unsuccessful, and therefore we have only weak

evidence for its role in viralreplication.

Inthis report we describe the construction ofa cell line which can express HSV-1 gDin an inducible fashion. The

cell linewasusedtoisolatea mutantHSV-1inwhichthegD structural sequences werereplaced by the Escherichia coli

,-galactosidase

gene. Virus

particles lacking gD

were

pro-duced innoncomplementingVerocells, and these particles

were able to bind to cells but not to initiate an infection. Therefore, gD is essential for penetration of HSV-1 into cells,aprocesswhichappears tobemore

complicated

than that previously described for small RNA viruses such as

vesicular stomatitis virus andinfluenza virus.

MATERIALS ANDMETHODS

Cells and viruses. Vero cells were grown in a minimal

essential medium (a-MEM) (GIBCOLaboratories,

Burling-ton, Ontario, Canada) supplemented with 7% fetal calf

serum (FCS). VD60 cells were maintained in Eagle MEM

lacking

histidine

(MEM-his)

supplemented with 0.3 to 1.2 mMhistidinol(Sigma Chemical Co., St.Louis, Mo.)and7%

FCS. Prior to infection, VD60cells were passaged at least onceina-MEMcontaining7% FCS. HSV-1 F and HSV-2 G wereobtained fromP. G. Spear, Northwestern University, Chicago, Ill., and were propagated and assayed by plaque

formation on Vero cells. The recombinant virusF-gD, was propagated and assayed on VD60 cells.

Plasmids. PlasmidpSS17 contains a copy of theBamHI J

fragment derived from HSV-1 KOS inserted into pUC19.

pMC1871 (5) was agiftfrom M. J. Casadaban. pTZ19R was

purchased from Pharmacia, Dorval, Quebec, Canada. pSV2HIS containing the Salmonella typhimurium histidinol

dehydrogenase gene under control of the simian virus 40

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HSV-1 GLYCOPROTEIN D MUTANT 1487

earlypromoter and flanked by simian virus 40 polyadenyla-tion sequences was a generous gift of R. A. Weinberg,

Whitehead Institute, Cambridge, Mass., and will be

de-scribed in more detailelsewhere (S. C. Hartman and R. J.

Mulligan, manuscript in preparation). Plasmid pSV2HISgD contains the BamHI J fragment of HSV-1 derived from

pSS17 and inserted into theBamHI site ofpSV2HIS. Antibodies.The HSV-1 gD-specific monoclonal antibody, II-436, was a gift of P. G. Spear. Monoclonal antibodies 15IB32, which recognizes HSV-1 or HSV-2 gB, and 17aBl,

whichrecognizesHSV-1 and HSV-2 ICP6 (the large subunit

of ribonucleotide reductase) (2), were gifts from S.

Bac-chetti, McMasterUniversity. Polyclonalrabbitserum, made

in rabbits injected withcrystalline HSV-1 thymidine kinase

produced in E. coli, was a gift from W. Summers, Yale

University,New Haven, Conn.

Construction of VD60 cells. Subconfluent Vero cell mono-layers (100-mm dishes) were transfected with 20

jig

of

pSV2HISgD, using the CaPO4 technique (11), treated 4 h

laterfor60 to 70 swith mediumcontaining 15% glycerol and

7% FCS, andthenwashedtwice with medium. After 2 days theconfluentcellmonolayers weretrypsinizedand plated in 10 100-mm dishescontaining MEMminus hissupplemented

with 0.3 mM histidinol. Approximately 16 days later, indi-vidual colonies of cells were trypsinized by using steel

cloningcylinders and seeded in dishes containing selective medium. The transformants were screened by infecting

small dishes of cells with HSV-2, labeling the cells with

[35S]methionine,

and immunoprecipitating HSV-1 gD from

cell extracts by using HSV-1 gD-specific monoclonal

anti-body 11-436. A cell transformant, VD60, which expressed

high levels of HSV-1 gD when infected with HSV-2 was

chosen fromapool ofover 180transformants.

Transfection, marker rescue experiments, and selection of viruses expressing

3-galactosidase.

Infectious HSV-1 DNA was isolated from cytoplasmic nucleocapsids as described

previously (30) and cotransfected with appropriate plasmid

DNAbytheprocedure ofGraham and van der Eb (11). At 4 hafterthe DNA wasadded, cellmonolayersweretreated for 60 swith mediumcontaining 7% FCSand 15% glyceroland washedthree times with medium. Viralcytopathiceffect was

observed2to 4dayslater, and the cells wereharvested and

sonicated. Inmarker rescue experiments, virusyields were

assessedinplaqueassayswithVeroand VD60 cells.

Recom-binant viruses abletoexpress,B-galactosidase were isolated by infectingVD60cell monolayers andoverlayingthe

mono-layers36 to 48h laterwith medium containing 0.5% agarose, 5% FCS, and 300 ,ug of

5-bromo-4-chloro-3-indoyl-P-D-galactopyranoside (X-Gal; Boehringer Mannheim Canada Ltd., Dorval, Quebec, Canada) per ml. Blue plaques

ap-peared after18to 40 h.

[35S]methionine

labeling ofcells, immunoprecipitation,and

gelelectrophoresis. Monolayers ofVero cells or cells

trans-formed with

pSV2HISgD

in35-mmdisheswereleft

uninfec-ted or wereinfected with HSV-1 or HSV-2. After2to 3 h, the cells were labeled with

[35S]methionine

as described previously (13). Cell extracts were madeat 7to 8 h postin-fection,andextracts were sonicated, clarified by

centrifuga-tion,andmixedwith mouseascitesfluidor rabbit serum and

protein A-Sepharose asdescribed previously (13). Samples

ofprecipitated proteinswereelectrophoresed in8.5%

N,N'-diallytartardiamide cross-linked polyacrylamide gels,

in-fused with2,5-diphenyloxazole, dried, andexposed to XAR film (Eastman Kodak Co., Rochester, N.Y.) as described previously (13).

Purification of labeled virions and adsorption of virions to

cells. Virions were purified from Vero orVD60 cytoplasmic

extracts by usingdextran-T1O (Pharmacia) gradients essen-tially as previously described (16, 32), except that labeling took place from 3 to 24 hpostinfectionin medium 199lacking

methionine and supplemented with 2% FCS and 20 p.Ci of

[35S]methionine per ml and fewer cells per gradient were used (2 x

107

to 4x

107

cells pergradient). To collect labeled virus, peak fractionscontaininglabeled virions were pooled, diluted 10-fold in 1 mMphosphatebuffer, and centrifuged at

4°C for2 h at 25,000 rpm in aTi5Orotor(Beckman Instru-ments, Inc., Fullerton, Calif.). The pelleted virus was

sus-pended, Dounce homogenized briefly, diluted in medium, and storedat 4°C.Replicate culturesofVero cells(orhuman

R970-5 cells in other experiments) in 24-well dishes were exposed to radiolabeled virus diluted in medium containing

2%

FCS at 37°C, and at appropriate times the medium

containing unadsorbed virus was removed, the cells were washed twice, and cell extracts were made by using

Tris-saline (50 mM Tris hydrochloride (pH 7.5), 100 mM NaCI) containing0.5% sodium dodecyl sulfate. Fractions contain-ing unadsorbed virus, washes, and cell extracts were indi-vidually dried on glass fiber filters and counted by liquid

scintillation spectrophotometry.

Electronmicroscopy.At20 hpostinfection, Vero or VD60 cells infected with F or

F-gDP

were washed and fixed at roomtemperature with 0.1 Msodium cacodylatebuffer (pH 7.2) containing 2%gluteraldehyde. The cells were scraped

fromthe dishes, collected bycentrifugation, andprocessed

forelectron microscopyasdescribed previously (15).

Polyethyleneglycolenhancementof viruspenetration. Vero cells were infected with F or

F-gDp

for 2 h, washed three times with medium containing 1% pooled human immuno-globulin and once in medium without gamma globulin and

harvested immediately or after 24 h. Samples of

virus-infected cells were sonicated, diluted appropriately, and

incubated with VD60 cell monolayers for 2 h at 37°C. The

cells were exposed briefly to polyethylene glycol, washed, and incubated as described previously (29). Plaques were

countedafter 48 h.

RESULTS

Construction of a cell line able to express HSV-1 gD and complement mutant viruses.Ontheassumptionthat gD is an

essential viral polypeptide, we constructed a cell line

con-taining endogenous gD gene copiesthat are expressed fol-lowing HSV infection. Vero cells were transfected with

plasmid pSV2HISgD, which containstheBamHI J fragment

ofHSV-1 (including the gD gene) inserted intothe BamHI

site ofpSV2HIS, avectorcontainingthehistidinol dehydro-genase genefromS.typhimurium under control of the simian virus 40earlypromoter (HartmanandMulligan,in

prepara-tion). Cells expressinghistidinol dehydrogenase areable to grow in medium containing histidinol and lacking histidine (an essential amino acid for most cells) by catalyzing the

oxidation ofhistidinol(toxicto mostcells)tohistidine. Cell

transformants able to grow in mediumcontaining histidinol

andlacking histidine were screened by infecting small dishes

ofthecells with HSV-2 and

immunoprecipitating

the HSV-1 gD expressed from endogenous HSV-1 gD gene copies by

usingmonoclonal antibody II-436, whichspecifically recog-nizes HSV-1gD and not HSV-2 gD. One cell transformant,

denoted VD60, was found toexpress high levelsofHSV-1 gD after infection with HSV-2(Fig. 1). All of the other 180 cell transformants screenedexpressedundetectable levelsor

muchlower levels ofgD than did VD60 cells and Vero cells

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1488 LIGAS AND JOHNSON

HSV-1

51 52 53

59 60 61

S

SF

-pgD

-gD

To confirm that the defect in replication of

F-gDP

results fromdisruption of the gD gene and not disruption of thegI

gene, weperformed a series of marker rescue experiments. VD60cellsweretransfectedwithF-gD, DNA andplasmids containing an intactgD and gIgene (pSS17), an intactgD geneand adisruptedgIgene(pUS7kan),or an intactgI gene

anda disrupted gD gene (pUS6kan). The titer of the virus

from these transfections was determined using VD60 and Verocells. Plasmids containing an intactgDgene, coupled

toeitheran intact (pSS17) ordisrupted (pUS7kan) gIgene,

efficiently rescued the

F-gDP

mutation, whereas plasmids containing a disrupted gD gene (pUS6kan) or no gD gene

FIG. 1. Expression of HSV-1 gD in Vero cell transformants infected with HSV-2. Cell lines VD51, VD52, VD53, VD59,VD60, and VD61 were derivedafter transfection of Vero cells withplasmid pSV2HISgD and selecting for cells abletogrowinmedium contain-ing histidinol andlacking histidine. Thetransformantswereinfected with HSV-2 and labeled with [35S]methionine. For comparison, Vero cells were infected with HSV-1 and labeled (lane HSV-1). HSV-1 gD was immunoprecipitated from extracts of the cells by using monoclonal antibody11-436,which doesnotrecognize HSV-2 gD.

infected with HSV-1. Insubsequentexperiments, wefound

that VD60 cells contain

approximately

110 copies of pSV2HISgD and donot express detectable amounts ofgD priortoinfection(data notshown),presumably becausethe gDpromoteris responsive toviral trans-activating factors.

VD60cellsmaintained the propertyofinducibleexpression ofgD afterHSV-2 infection for atleast 20 passages in the

absence of selection. We also found that the inducible expressionofgDincreasesifthe cellsaregrownin medium lacking histidine and containing 1.2 mM histidinol for 12 passages (data not shown). The rate ofdoubling of VD60

cellsin selectiveornonselective mediumissimilartothatof

Vero cells in nonselective medium. We found previously thatthegrowthratesofneomycin-resistantor

methotrexate-resistant transformants were significantly reduced in

selec-tive media.

Construction ofavirusmutant,HSV-1

F-gDO,

in which the gD gene is replaced by theE. coli

jI-galactosidase

gene. To

isolate a mutant virus lacking the gD gene, we first

con-structed a plasmid, pDGAL11ZK, in which the sequences

encoding thegDpolypeptide andpart oftheneighboring gI

gene were replaced by E. coli

P-galactosidase

sequences

fused togD promoter elements (Fig. 2). VD60 cells were

transfected with pDGAL11ZK and intact HSV-1 F DNA,

andvirusesderivedfrom thetransfectionwere screened by

overlaying plaques formed on VD60 cell monolayers with

agarose containing X-Gal. A recombinant virus,

F-gD3,

whichproduced blue plaques under X-Gal overlay (Fig. 3a), was isolatedand plaque purified.

Southern

blot analysis of

F-gD,B DNA (data not shown) indicated that the virus

contained,-galactosidasesequences inserted inplace ofgD andgI structural sequences (Fig. 3b).

Phenotype of the mutant virus and marker rescue with

plasmids containing the gD gene.

F-gDP

formed plaques on VD60 cells which were of normal size, but was unable to

formplaquesonVerocellsorhuman R970 cells, suggesting

that some stage of the replicative cycle of the virus was

disrupted. The substitution mutation disrupts both the gD and gI genes; however, the gI gene has been shown to be

dispensablefor HSV-1 replication in cultured cells (14, 18).

FIG. 2. Construction ofplasmid PDGAL11ZKusedto mutage-nize the HSV-1gD gene. Plasmid pSS17, which contains theBamHI

Jfragment of HSV-1 insertedintopUC19,wasdigestedwithNcoI,

treated with the Klenow fragment of DNA polymerase I, and digested withSmaI,andafragmentcontainingthegD promoterwas

purified and inserted into the SmaI site of pMC1871 directly upstreamof the

0-galactosidase

structural sequences.APstI

frag-mentcontaining the gD-p-galactosidase hybrid genewas excised frompDGAL11 and inserted into thePstIsite of pTZ19R. HSV-1 sequences 3' tothegD gene, extending fromauniqueBalI site in pSS17 toaBamHI site, were excisedfrom pSS17K (aplasmidin

which the BalI site in pSS17 was converted to a KpnI site) and inserted downstream of theP-galactosidasesequencesat aKpnIsite in pDGAL11Z to form pDGAL11ZK. Abbreviations: B, BamHI;

B1, BalI; K, KpnI; N, NcoI; Ps, PstI; S, SmaI; S1, SaII; Apr,

ampicillin resistance gene;Tcr,tetracycline resistance gene; ,-gal.,

P-galactosidasesequence.

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a

UL~~~ ~ ~ ~ ~ ~ ~ ~~

~~'-~~~-~'~

H N F Bal

_

...

HSV-1(F) USBIgo) Us7(gh Us81gE1

B H

HSV-1(F-gDL) 3-Galactosidase Us8(gE)

FIG. 3. Expression of ,-galactosidase by the recombinant virus

F-gD,B and schematic representations oftheDNA sequence arrange-mentsin wild-type HSV-1F and mutant strainF-gDf3.(a)VD60cells were infected with HSV strain F (right) or

F-gDP

(left), and after 36 h the monolayers were overlaid with medium containing agarose and

X-Gal. Blue plaques appeared after 36 h. (b) The region of HSV-1 DNA containing the US6 (gD), US7 (gI), and US8 (gE) genes is

depicted for HSV-1 F and recombinant

F-gDP

(21). In

F-gDP

P-galactosidase

sequencesreplacegDstructural sequences and part

ofthegIgenefromanNcolsiteneartheinitiationcodonofgD to a

BalI site in thegI gene. Abbreviations: B, BamHI; Bal, BalI; H,

HindIll; F, FspI; N, NcoI; UL, long unique segment; Us, short

uniquesegment.

(pUC19)wereunable to rescue

F-gDP

(Table 1). Therefore, the lossof the gD gene and not the gI gene leads to a defect in the replication of F-gD, on Vero cells. We find that viruses with disruptions in the gI gene replicate to titers equal to that of wild-type viruses (14; D. C. Johnson,

un-published results).

Formation of noninfectious virus particles by

F-gDIO

in noncomplementing cells.Although

F-gDP

was unable to form plaques on Vero cells, Vero cells displayed typical HSV cytopathic effects when the cells were infected with a high

multiplicity (5 to 10 PFU per cell) of F-gD,3 grown on

complementing VD60 cells. This suggested that

F-gDP

vi-TABLE 1. Marker rescue of F-gD,Bby plasmidscontaining

intactordisruptedgD andgIgenes Titerb

Plasmid' (Vero/VD60)x 100%

VD60cells Verocells

pSS17 8.2 x 106 4 x 105 4.9

pUS6kan 8.0 x 106 <103 <0.013

pUS7kan 2.0 x 106 8.2 x 104 4.1

pUC19 2.4 x 107 <103 <0.004

"VD60 cells were transfected withF-gDj3 DNAand one of the plasmids indicated. pSS17 containstheBamHI Jfragment of HSV-1subcloned into pUC19; pUS6kanisidenticaltopSS17,exceptthat akanamycingene cassette isinserted intoaHindlIl siteinthe promoter of the gD gene; andpUS7kanis identicaltopSS17,except that akanamycingene cassetteis inserted into a BalI sitein thegIgene.

bTransfectionyieldsweremeasuredby plaqueformation on VD60 or Vero

cells.

rions grown on VD60 cells (and supplied with the gD polypeptide)wereabletoinitiate infection of Vero cells. To

testwhether

F-gDp

virionsgrown on VD60 cellswere able to complete the viral lytic cycle and produceprogenyvirions in Vero cells, weperformed two types of experiments. In the first set ofexperiments radiolabeled virus particles in cell extractswere quantitated by using dextran gradients. VD60 orVero cells were infected withF-gD,B or wild-type strain F and labeledwith[35S]methionine. Extracts from labeled cells were centrifuged on dextran T10 gradients (32), and labeled virions were quantitated. Peaks of labeled virions observed

in extracts fromn

F-gDp-infected

Vero cells and

F-gDp-infected VD60 cells were only slightly lower than those observed inextractsfromVero cells infected with wild-type strain F(Fig. 4). In the second set of experiments, electron micrographs of virus-infected cells revealed enveloped par-ticles in

F-gDp-infected

Vero cells which were structurally and quantitatively similar to those observed in

F-gDp-infected VD60 cells or F-F-gDp-infected Vero cells (Fig. 5). In

addition, weobserved numerous envelopedparticles near to and attached to the surfaces of

F-gDp-infected

Vero cells. Since Vero cells infected with F-gD, do not synthesize detectablequantities ofgD (seebelow), we conclude that gD is notrequired forassembly, maturation, or egress of HSV-1 virions.

Binding to cells of virus particles lacking gD. One hypoth-esis which might explain the inability of mutant viruses

bearing gD to form plaques on Vero cells is that gD is

required at a very early stage in the viral lytic cycle, i.e., virus adsorption to or penetration into cells. F-gD,B viruses

containing gD in their envelopes (grown on VD60 cells)

would be able to initiate a single round of infection on Verocells, but the progeny, lackinggD, would be unable to

initiate a second round of infection. To determine whether virions lacking gD could bind to Vero or VD60 cells, Vero cells were infected with F-gD, and labeled with

[35S]methionine,

and radiolabeled virus was prepared on

dextran gradients. Vero cells were incubated with labeled

28

24

20

16

12

U

..t

₅

L_.,.".4r

10 15 20 25 30

Fraction number

FIG. 4. Virus particles produced in cells infected with F-gDf3.

Vero cells or VD60 cells were infected with HSV-1 F or HSV-1

F-gD,p,

labeled with[35S]methionine, and harvested after20to24h,

and the virus particles were purified on dextran gradients. The trichloroacetic acid-precipitable label in gradient fractions was

quantitated,andonlyfractionscontainingradiolabeled virionswere

plotted. Symbols: 0, Vero cells infected with F; O, Vero cells infected with F-gD,;*, VD60 cells infected withF-gD,B.

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virus particles and washed and the label associated with the cell monolayer was quantitated. Virions purified from

F-gDp-infected

Vero cellswereabletobindtoVero cells and,

in other experiments, to human R970-5 cells (results not

shown) asefficientlyaswerewild-type Fvirions and F-gD, virions purified from VD60 cells (Fig. 6). Binding

experi-ments performed at 4°C gave similar results, although the

overallbinding of all viruses waslower(resultsnot shown). Therefore, gD is notessential for attachment orbinding of HSV-1 to cells.

Penetration into cells of F-gD,B virions lacking gD. To determine whether F-gD, virions lacking gD could initiate

certain of the initial stages of virus replication, namely induction of early viral polypeptides, Vero cells were in-fected with F-gDpderived from VeroorVD60 cells and the cells were labeled with [35S]methionine. HSV-1 early

poly-peptides, ICP6 (the large subunit of HSV-1 ribonucleotide reductase), thymidine kinase, gB,andgDwere

immunopre-cipitatedfrom cellextractsby using monoclonal or

polyclo-nal antibodies.

F-gDp

viruses containing gD in the virion envelope were able to induce the synthesis of viral early polypeptides ICP6, thymidine kinase, andgB (Fig. 7, lanes

3). However, these viruses cannot induce the synthesis of gD, as one would expect, because the gD gene has been deleted. In contrast,

F-gDP

viruses lacking gD (harvested fromVero cells)wereunabletoinduce the synthesis of viral earlypolypeptides (Fig. 7, lanes 2). Thisresultsuggeststhat virions lacking gDareunable topenetrate into cells.

To further examine the possibility that virus particles lacking gDare unableto penetrate intocells and initiate an infection,

F-gDP

virions lacking gD were incubated with

VD60 cells, and then thecells were treated with

polyethyl-ene glycol. Polyethylene glycol treatment has previously

been shown to facilitate the entry of gB mutants which

cannotpenetrateinto untreated cells (17,29). Inone

exper-iment, brief exposure ofVD60 cells infected with

F-gDP

virionslacking gD increased byover300-fold thenumberof

plaques formed (Table 2). In a second experiment, the polyethylene glycol enhancement ofplaque formation was lower,andwefound that

F-gDP

harvested2 hafterinfection of Vero cellsproduced approximately2x 104PFUonVD60

0

u

0

-1

.0

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Time(minutes)

FIG. 6. Binding of purified viral particles lacking gD to cells. Virus particles labeled with [35S]methionine were purified on dex-tran gradients from Vero cells infected with F (0). Vero cells infected with

F-gDP

(OI),orVD60 cells infected withF-gD,3 (M)and

incubatedwith Verocellsat37°Cin mediumcontaining2% FCS. At various times the cells were washed and radiolabel bound to the cells was quantitated. The results shown are the average oftwo

independent experiments.

ICP6 tk gB gD

1 2 3 1 2 3 1 2 3 1 2 3

97K-

68K-ICP6 gB

-pgB

* gD

a

-pgD

-tk

46K-FIG. 7. Expression of early viral polypeptides in cells infected with HSV-1 F or HSV-1 F-gD,B. Vero cells were infected with HSV-1 F harvested from Vero cells (lanes 1), HSV-1

F-gDp

harvestedfromVerocells(lanes 2), orHSV-1F-gDpharvested from VD60 cells (lanes 3). The cells were labeled with [35S]methionine from2to 6hafterinfection, and then cell extracts were mixed with monoclonal antibody 17aBl specific for ICP6, rabbit antibody specific for thymidine kinase (tk), monoclonal antibody

15PB5

specific for gD,orantibody 11-436 specific for gD. The immunopre-cipitated proteinswere eluted andelectrophoresed on sodium

do-decylsulfate-polyacrylamide gelsandsubjectedtoautoradiography. Molecularmassstandards areindicatedattheleft.

cellmonolayers.Thissuggeststhat therelatively high

back-grounds in these experiments result from residual F-gD, virionscontaining gD which were used to prepare stocks of virus in Vero cells.

DISCUSSION

HSV-1 gD is a majorcomponent ofthe virion envelope

andinfectedcellmembranes,andvirusneutralizing antibod-ies oftenrecognizegD (7, 12a, 22,26). Numerous attemptsto

isolate viruses with mutations in the gD gene have been unsuccessful. These results suggested that gD might be an

essential component of the HSV-1 envelope or carry out someessentialfunctionduringthereplication cycleof HSV-1. Assuming thatgD is essential for HSV-1 replication, we

beganourattemptsto mutagenizethegDgeneby

construct-ingacellline able tocomplementviruses withmutations in thegD gene. Cellswere transfected withaplasmid

[image:6.612.59.297.503.652.2]

contain-ing the gD gene coupled to a novel selectable marker,

TABLE 2. Plaque production of F-gD, particleslackinggD afterpolyethylene glycolenhancement

Time(h)

Titerb

~

+E/E

Virus harvested' EG G +PEG/-PEG

Expt1

F 24 1.3 x 109 8.3 x 108 1.57

F-gD, 24 6.3 x 106 2.0 x 104 315

Expt2

F 24 5.2 x 108 3.4 x 108 1.53

F-gD, 2 3.0 x 104 2.0 x 104 1.50

F-gD,3 24 1.8 x 106 1.5 x 104 120 "Vero cells were infected with F or F-gD,B, and infected cells were harvestedatthe indicatedtimes.

bVirus-infected Vero cellswere sonicated, and virus titers were deter-mined onVD60cellswithorwithoutpolyethylene glycol(PEG)treatment.

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histidinol

dehydrogenase,

andtransformants abletogrowin

medium

containing

histidinol and lacking histidine were

isolated.

We

found that this selection

system

offeredsome

advantages

overother available methods

involving

neomy-cin or

G418, hygromycin,

and methotrexate

resistance,

whichareoftenmoretoxictotransformantsor more

expen-sive.

Although

many of our histidinol-resistant

transfor-mants

expressed

detectableamountsof

gD, only

oneofthe 180 transformants

screened, designated VD60,

was able to

express

gD

at levels

comparable

to those observed in in-fected cells.

To assess the effects of

deleting

the

gD

gene on virus

replication

and,

for future

studies,

to allow us to

rapidly

select forviruses in which the

gD

gene hasbeen

mutagenized

in

vitro,

we

replaced

the

gD

gene and a

portion

of the

dispensable

gI

gene with the E. coli

P-galactosidase

gene.

Recombinant viruses

expressing ,B-galactosidase

were

readily

selectedonthebasis of their

ability

to

produce

blue

plaques

under

agarose-X-Gal overlays. Preliminary

experi-ments also indicate that we can select for viruses which

acquire, by

marker

transfer, copies

of the

gD

gene

contain-ing

site-directed mutations

by isolating

viruses which

pro-duce colorless

plaques.

In

addition,

we can

readily

screen

DNA sequences essential for

gD

function

by using

marker

transfer

experiments

suchasthose described inTable 1.

The mutant virus

F-gD,

was able to

replicate

on VD60 cells, butwasunableto

complete

afull round ofinfection in

Vero cells. Normal amounts of virus

particles

were

pro-duced

by

themutantvirus in Verocells inwhich

gD

is not

expressed.

These virions

appeared structurally

identical to

wild-type

virions, although

amore detailed structural

anal-ysis, perhaps involving negative staining

(33),

would be

required

toreveal smalldifferencesin

envelope

structure. In

addition, F-gDP

virions were

distributed

throughout

the

cytoplasm

and on the surfaces ofVero cells. These results

show that

gD

is not essential for HSV-1

envelopment,

maturation,

oregressto thecell

surface.

Previous studies have

suggested

that gD

might

be an

essential component in the attachment of virus to cell surfaces. Virosomes or

lipid

vesicles reconstituted with virion

glycoproteins

were able to bind to

cells,

and the

binding

was reduced when

gD

or

gB

was removedfromthe

lipid

vesicles

(16).

In

addition, polyclonal

and monoclonal

antibodies

specific

for

gD

inhibited

the

binding

of virionsto

cells

(9).

In the studies

reported here,

virus

particles

in which

gD

was

completely

absent from thevirion

envelope

wereabletobindto

monkey

orhumanfibroblastsaswellas

virions

containing gD

were.

Therefore,

itappears thatgD is alsonotessentialfor virus

attachment,

atleasttothesecells.

Itis

conceivable,

however,

that

gD plays

anessentialrolein

HSV attachment to other cell types, for

example

neurons.

We

plan

to testthis

possibility using

virusmutants.

Initially,

wefound it somewhat

surprising

that

gD

doesnot

play

an essential role in virus

binding

to cultured

cells,

because there is now some evidence thata

majority

ofthe

other HSV-1

glycoproteins

are also not essential for virus

attachment to cells or virus

replication.

Virus mutants

un-abletoexpress

gB (S. Person, personal communication),

gC

(12, 28), gG, gE,

or

gI

(14,

18,

19,

23,

37)areabletobind to and in some cases

replicate normally

incultured cells. The

simplest

explanation

for

these

results isthattwoor moreof

these surface

glycoproteins

can

independently

mediate the

initialattachment of virus tothecell surface. Inadditionto

gD, gB

wouldseem tobea

likely

candidate for membership

in this group ofcell attachment

proteins, because

notonly

did virosomes

depleted

of

gB

bind poorly to cells (16) but

also heparin binds to

g1B

(M. L. Parish and P. G. Spear, unpublished results)andblocks virusadsorptiontocells(35, 36). There is also evidence that gC and gEmay be involved in virusbinding tocells(8).

Althoughable to bind to cells, virusparticles lacking

gD

wereunableto initiate the synthesis of early viral polypep-tides, suggesting that either entry of virions into cells or some subsequent stage in virus replication was blocked.

Polyethylene glycol treatment dramatically increased the

plaque production of

F-gDP

virionslacking gD on comple-menting VD60 cells.These resultssuggestthat HSV-1gDis

essential for virus penetration into cells. However,

F-gDP

virions harvested from Vero cellsaredeficient ingI aswell

as gD. AlthoughgI has been shown to be dispensable for

virusreplication (14, 18)and aplasmid containing anintact

gD gene and a disrupted gI gene efficiently rescued the

replication

defect in

F-gDp,

it ispossible that gI somehow

affects the phenotype of the

F-gDP

virions, perhaps by interacting with gD. We think it unlikely that gD and gI

interact,

because gI complexes with gE to form IgG Fc receptors,whicharedispensable forvirusreplication (14).In

addition, we have recently characterized virus particles whichcontaingIbut notgD, andthesevirusesareunable to

infectVero cells (M.W. Ligas and D. C. Johnson, unpub-lishedresults).

There is evidence that gB, aswell as gD, is involved in

viruspenetration into cells (17, 29). Monoclonalantibodies to gH prevent virus-induced cell fusion (10), a process

thought

tobeanalogoustovirus entryinto cells,andviruses

lacking

gH arenoninfectious (Desaietal., Abstr. 12th Int.

Herpesvirus Workshop); thus,

gH mayalso be involved in virus

penetration.

One might suggest that gD, gB, and gH

molecules froma complex which acts as the virus

attach-ment

component

and subsequently induces membrane fu-sion.

However,

there is evidence that gB and gD are

spatially separated

in the virion envelope (33) and thatgB forms homodimers which are not associated with other

glycoproteins

(6). A more likely hypothesisis thatmultiple interactions occur between individual HSV glycoproteins andanumberof cell surfacecomponents, sothatthevirion

envelope

is

brought

into close proximity to the plasma

membrane and the twomembranes areinduced to fuse. In

this

model,

gD, gB,orgHmay notbe essential fortheinitial

binding

of virusto

cells,

butmaybe essential for interactions whichsubsequently lead toviruspenetration in additionto

being directly

involved in the membrane fusion reaction. Fusion may involve conformational changes in the fusion

polypeptide,

aswith the

pH-induced

conformational change

inthe influenza virushemaglutinin,and mayberegulated by other

polypeptides.

HSV-induced fusion of infected cells, most easily

ob-served with

syncytial

strains of

virus,

is thought to be a processanalogoustofusion ofthevirionenvelope with cell

membranes during virus entry into cells (31). Antibodies

directedto gD, as well as to gH, block virus-induced cell fusion(10, 12a, 24). Wefound thatF-gD,Bcausedrapidcell

fusion ofVD60 cells, but did not fuse Vero cells (datanot

shown).

Thisresult

implies

thatgDplaysanessentialrole in cell-cellfusion inadditiontoviruspenetration.

It is noteworthy that the gD gene, here shown to be essentialfor virusreplication, isembedded in a cluster of 12 geneswhich makeup the unique S componentofthe virus

genome (21). Eleven of these genes are dispensable for

replication

of HSV-1 in cultured cells (14, 18, 19, 23, 37). The

observationthat the

majority

of

S-component

genescouldbe

deleted ledLongneckerandRoizman(19) to suggest that the

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S component was acquired as a DNA fragment which subsequently evolved to allow the virus to survive in its environmental niche in the human host. If this hypothesis is

correct, the ancestral herpesvirus did notdependon gDto

enter cells. This scenario predicts alterations in the entry

pathway of HSV following acquisition of the Scomponent.

Alternatively, one might hypothesize that the genes sur-rounding gD have playedanimportant role in the replication of HSVthroughoutmostof the evolution of the virus andyet

thesegenes aredispensable in the limited setting of

labora-tory-cultured cells. There is evidence suggesting that

S-component genes confer a distinct selective advantage on the virus in the human host. We haverecently shown that S-component polypeptides gI and gE formacomplex which acts as an immunoglobulin G Fc receptor and may act to

shield virus-infected cells from the host immune response

(13, 14). Additionally, thegeneencoding gG, whichmapsto

the left ofgD (1, 27), has been implicated in virus replication inthe central nervous system(37). From this perspective it is not surprising that the gD gene is surrounded by genes

encoding functions which are advantageous and may be

essentialfor virus replication in humans.

ACKNOWLEDGMENTS

We are indebted to James Smiley for coaching, especially in matters havingtodo with genetics. We thankSteven Hartman for

suggestingtheuseofpSV2HISand PatriciaSpear,SilviaBacchetti,

and William Summers forgiftsofantibodies.JosephinaMaljarwas

invaluable inhelpingtoputthemanuscript together.

This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. D.C.J. is a research scholar of the National Cancer Institute of Canada.

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