Cell lines containing varicella-zoster virus open reading frame 62 and expressing the "IE" 175 protein complement ICP4 mutants of herpes simplex virus type 1.

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0022-538X/88/062076-07$02.00/0

Cell Lines

Containing

Varicella-Zoster

Virus

Open

Reading Frame

62 and

Expressing

the

"IE"175

Protein

Complement

ICP4

Mutants

of

Herpes Simplex

Virus

Type

1

JAMES M.

FELSER,1

PAUL R. KINCHINGTON,2 GENEVIEVE

INCHAUSPE,1

STEPHEN E. STRAUS,'

ANDJEFFREY M. OSTROVEl*

Medical Virology Section, Laboratory ofClinicalInvestigation, NationalInstitute of AllergyandInfectious Diseases,' and the

Unifrnrmed

Services

University of

the Health

Sciences,2

Bethesda,

Maryland

20892

Received 7 October1987/Accepted22 February 1988

Vero cellswerecotransfectedwithpSV2neo andarecombinantplasmid containingthe varicella-zostervirus (VZV) open reading frame 62 (ORF62). Three neomycin-resistant cell lines were isolated and shown to complement two different ICP4 mutants (tsB21 and d120) ofherpes simplex virus (HSV) type 1 (HSV-1).

VZV-specific RNA could notbe detectedinthese celllines, butfollowinginfection with tsB21,a4.3-kilobase VZV transcript was detected. This RNA increased in quantity when cells were infected in the presence of cycloheximide.AVZV-specific proteinof 175 kilodaltonswasdetected in extracts of all three cell linesfollowing infection with wild-type HSV-1 butnotinuninfectedcells. That VZV RNA andproteinweredetectedonlyin HSV-1-infected cellssuggeststhatacomponent of the HSVvirion activates theexpressionof VZVORF62.The increase in RNA expressionseeninthepresenceofcycloheximideindicates that theproteinencodedbyVZV

ORF62, "IE"175, may beautoregulatory. These data provide further evidence that VZV "IE"175 is the functional analog of the HSV ICP4.

Expression of many herpesvirus genes is coordinately regulatedandsequentially orderedinacascade fashion. The mostextensive analyseshave been done forherpes simplex virus (HSV) type 1 (HSV-1) genes, which are divided into

five temporal groupings (ox,

2,

yl, and "Y2). Five cx or

immediate-early genes of HSV are known. These are

ex-pressed first ininfection and aretranscribed in the absence of de novo protein synthesis (20, 21). The HSV and y

classes of genes are expressed as early and late genes,

respectively.

Less extensiveanalyses ofthetemporalexpressionof the genesofotherherpesviruseshave been conducted. No such studies have been performed with varicella-zoster virus

(VZV), in part because of the difficulty in obtaining high

titers of cell-free virus requiredto synchronously infect the

large numbers of cells needed to study gene expression. Nevertheless, among the products of the 71 open reading frames (ORFs) predicted by theVZV DNAsequence, there are a number ofpolypeptide homologs between HSV and VZV (11). Fromthese, Davison and Scott (11) have

identi-fiedhomologsin VZVfor three HSVotgenes(ICP4, ICP22,

and ICP27) (11). The predicted product of VZV ORF62

sharessignificant amino acid homology with the HSV ICP4. Three regions of the predicted VZV protein, representing approximately650amino acids,haveover50% homologyto

ICP4(10).TheORF62 product is predictedtobeatleast 140

kilodaltons(kDa) in size (10, 11)compared with175 kDa for

the HSV-1 ICP4 (36). Recently, Shiraki and Hyman (29)

detected four immediate-early proteins of VZV. The one

theyestimate to be 185 kDa is closest in sizetothe protein

predicted forORF62. We reported previously that a DNA

segment containing VZV ORF62 complements an ICP4

temperature-sensitive (ts)mutant ofHSV-1, tsB21, thereby

mappingan ICP4-equivalent activity toORF62 (16). In this report, we describe the establishment of

trans-* Corresponding author.

formed cell lines which contain andexpresstheORF62gene

of VZV. These cell linesefficiently complementtwodifferent

ICP4 mutants of HSV-1. Under the conditions at which

ORF62isefficiently expressedin thesecells, we can detect and characterize its transcriptand a 175-kDaprotein prod-uct.

MATERIALS ANDMETHODS

Cells and viruses. Human embryonic fibroblasts (Flow 5000), human fetal foreskin fibroblasts (HFFs), and CV-1 and Vero cellswere propagatedina 1:1 mixtureof minimal essential medium and medium 199 supplemented with 10% heat-inactivated fetal bovine serum, 2mM glutamine, peni-cillin G (100U/ml), andstreptomycin (100 ,ug/ml). E5 cells,

agiftof NealDeLuca,containthegeneencodingthe HSV-1 ICP4 and are able to complement HSV-1 ICP4 deletion

mutants(30). TheKOS strain of HSV-1 andtsB21,an ICP4

temperature-sensitive mutant of HSV-1 (KOS), were

pro-vided by Priscilla A. Schaffer (14). HSV strain d120, also

courtesy of N. DeLuca,isamutantof KOS which contains a4.1-kilobase (kb) deletionin thegeneencoding ICP4 (13). VZV strains Ellenand Scottwere grown in Flow 5000 and HFFcells, respectively, aspreviously described (35).

Plasmids. pVZVEcoE contains the EcoRI E fragment (Fig. 1)of VZV strainElleninpBR325. pVZVEcoA contains

the EcoRI A fragmentof VZV strain Ellen in pBR322 (9). pGI26 (Fig. 1)contains the 6.5-kbBglII-HpaI fragment from pVZVEcoA ligated into the SmaI site of pUC9 following blunting of ends with T4 DNA polymerase. Plasmid

pSV2neo contains the bacterial gene encoding G418 resis-tanceunder thetranscriptional controlofthesimianvirus 40

early promoter (32). pSG1, courtesy ofSandra K. Weller, containsthe EcoRIJKfragment, includingthe ICP4geneof

HSV-1 cloned into pBR325 (18).

Nucleicacidisolationandhybridization.CellularDNAwas

isolated and hybridized as described previously (28, 31). Total cellularRNA was isolated and purified by the

guani-2076

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MAP UNITS

vzv

0.7 0.8 0.9

a a a

1.0

UL IRL IRS U TRS

*

I

4.3Kb* 4.3Kb

E

FIG. 1. The mapposition of ORF62 is shown in the right-hand

portion of the VZVgenome. PlasmidpGI26 contains the indicated

BglII-HpaI6.5-kb fragment cloned into pUC9. Transcript mapping datahaslocalizeda4.3-kb transcripttoORF62asshown previously

(26). Thelocations of restriction sites forenzymesEcoRI (E),BglII

(B), and HpaI (H) arebasedonpublished sequence data (11). UL

andUs, Unique long and shortsegmentsof the virus;IRSandTRs,

internal and terminal repeats of the short segment; IRL, internal repeatof thelongsegment.

diniumthiocyanate-cesium chloride procedure and

hybrid-izedas previously described (7, 17, 26).

Complementationof tsB21byclonedDNAfragments. Com-plementation of tsB21 by protein expressed transiently from transfected DNA fragments was carried out by using a modificationof theprocedure of Sacksetal.(27).CV-1cells

were seeded into 60-mm-diameter dishes (6.25 x

105

cells per dish). The next day, the cells were transfected in

duplicate with0.5 ,ugofplasmidDNAand 10 jig ofcarrier DNA (salmon testes) in 0.5 ml of HEPES (N-2-hydrox-yethylpiperazine-N'-2-ethanesulfonic acid)-buffered saline. Four hours later, cells were treated with 15% glycerol in HEPES-buffered salinefor 2min.Thecellswereinfected 24 hlaterwithtsB21at39°C atamultiplicityof 2 PFUpercell. After incubation for 20 h at 39°C, the cultures were har-vested and virus titers were determined by the amount of

plaqueon Verocells at34°C.

Viral complementation studies. Virus titrations were per-formed on various cell lines as previously described (16).

Time course complementation studies were carried out by seeding the indicated cells into 60-mm-diameter dishes in

duplicate. The nextday, cells were infected with d120at a

multiplicity of 2 PFUpercell andwere maintainedat37°C.

Cultureswereharvestedatvarious times after infection.The yieldof d120wasdeterminedon E5 cells at37°C.

Biochemical transformation of Vero cells with plasmid

DNA. Vero cells were cotransfected with pSV2neo and

pGI26,andG418-resistant colonieswereselected andgrown

by using a modification of the technique described by

DeLuca etal. (13). Briefly, unlinearized pSV2neo (0.5 ,g)

and pGI26(20

R,g)

werecoprecipitated in the presence of 40

,ugof salmon testes DNA in a total volume of 1.0 ml by the calcium phosphate procedure of Graham and van der Eb (19). Theprecipitated DNA was then added to subconfluent monolayers of Vero cellsin60-mm-diameter dishes. After a 4-h incubation at 37°C, the cells were shocked with 15% glycerol andincubated for an additional 2 days at37°C. The cells were thentrypsinized and seeded onto 85-mm-diameter

dishesatadensity of about 5 x 103cells percm2. After cell

attachment, the medium was removed and replaced with

medium containing 1 mg of active G418 per ml. After a

further 5 to 10 days, the G418 concentration was lowered to

600 ,ug/ml. At 12 to 18 days after transfection, individual

G418-resistant colonies wereisolated, amplified, and

subse-quently maintainedin the presence of G418 (600 ,ug/ml).

Western blot (immunoblot) analysis. Rabbit antisera were prepared against a synthetic peptide corresponding to the

C-terminal12amino acidspredictedfrom the sequence data

forVZVORF62(11; P. R. Kinchington, W. Ruyechan, and

J. Hay, manuscriptinpreparation). The peptide was

conju-gated to keyhole limpet hemocyanin and injected into

rab-bits, whereupon antisera were derived. A peptide

corre-spondingtotheC-terminal12aminoacids predictedforVZV

ORF29(22a), which has been identified as the VZV major

DNA-binding protein byhomology to HSV-1 (11), was also

synthesized.

A total of

106

cells were seeded into 60-mm-diameter

dishes in duplicate and infected on thefollowing day with

HSV-1strain KOSat37°Cat amultiplicity of2PFU percell.

The infected cultures were harvested 20 h later and the proteins were solubilized in 300

RI

ofbuffercontaining 2%

sodiumdodecyl sulfate, 5% 2-mercaptoethanol, 50 mMTris

hydrochloride, and5% glycerol (pH 6.7).

The proteins were separated by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis and electrophoretically

transferredtonitrocelluloseinbuffercontaining

0.25

MTris,

0.192 Mglycine, and 20% methanol. Following transfer,the

nitrocellulosewastreated with10% Carnation instant nonfat

milk inbinding buffer (1 mM EDTA, 10 mM Tris

hydrochlo-ride,0.15 MNaCl,0.02%Tween20[pH7.6]) for1h at25°C.

The

blots were then reacted with the anti-peptide sera

diluted 1:50 in 1% milk-binding buffer for 2 h at 25°C.

Unbound antibodies were removedby five5-minwashes in

binding buffer, and the blots werereacted with

"251-labeled

staphylococcus protein A (New EnglandNuclearCorp.) at

105dpm/ml in 1% milk-binding buffer for2 hat25°C. Free

protein A was removed by extensive washing in

binding

buffer. Autoradiography was performed on Kodak XAR5

film.

RESULTS

ComplementationoftsB21bycloned DNAfragments.

Com-plementationassayswerecarriedout todeterminethe

ability

ofHSY-1 ICP4or its

putative

VZV counterpartto

comple-menttsB21atthe

nonpermissive

temperatureof

39°C.

Plas-mid

pSG1,

which contains the ICP4 gene of HSV-1, and

pVZVEcoE and pGI26, which contain the VZV

ORF62,

were all found to

complement

tsB21 with

complementation

indices of40.4, 11.3, and13,

respectively

(Table

1).The

only

intact VZV ORF contained in pGI26 is

ORF62,

and this

plasmid was therefore usedto establish

stably

transformed

celllines.

Generation of cell lines

containing

ORF62.

pGI26

and

pSV2neowere cotransfected into Vero cells.G418-resistant

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colonies were isolated and screened for their ability to support the replication of tsB21 at 39°C. Three cell lines, termed F19, F113, and F114, efficiently supported the repli-cation oftsB21. Twenty hoursafter infectionwithtsB21ata

multiplicity of 2 PFU percell, cultureswere harvested and

their virus yieldswerecomparedwith thoseobtained

follow-ing infection of VN2 cells at 39°C. VN2 cells are a

G418-resistant line derived from transfection of Vero cells with

pSV2neo alone. Cell lines F19, F113, and F114 yielded, respectively, 26, 39, and 153 times more virus than did

infected VN2 cells. The titers ofthe virus recovered from

cell lines F19, F113, and F114were alsodeterminedat 39°C

onVero cellstodetect recombinantprogeny.Some celllines

cotransformed with the ICP4 gene of HSV-1 have been

showntoyield recombinantprogenyfollowinginfection with

tsB21 (8). Such cell lines contain an HSV-1origin of repli-cation. Recombinantscreeningwasperfortned followingour

experiments, because aVZV replicationorigin iscontained in pGI26 (34). No increase above background titer was

observed, suggesting thatthe efficientproductionof

recom-binantprogeny did notoccur.

We examinedEcoRI-digested DNAfrom the transformed cell lines andVero cellDNAbySouthern blothybridization analysis by using the gel-purified VZV EcoRI Efragmnentas a probe (Fig. 2). Of four cell lines derived from the initial cotransfection ofpSV2neo and pGI26, the three cell lines

which complemented tsB21 (FI9, F113, and F114) were found to contain VZV DNA sequences. The major band

noted is 9.2 kb, the size of linearized pGI26. Since intact

pGI26 is cut only once with EcoRI in the polylinker se-quence(Fig. 1),thedetectionofasingle majorbandsuggests

integration of the plasmid intothecellsas atandemrepeatof pGI26 at one or more sites. The presence of a number of bands smaller and largerthan 9.2kbmay reflectintegration

of VZV sequences into different areas of the cellular

gen-ome.Theintegrationpatternisuniquefor eachcell line. One cell line (FIll) which did notcomplement tsB21 was nega-tive for VZV sequences. Two other cell lines(F16.and F17)

contained VZV sequences but have not been tested for

abilitytosupporttsB21 replication.VN2cellswerenegative forpGI26sequences, aswere Vero cells.

Cell lines containing ORF62 complement ICP4mutants of

HSV-1. Since initial screening demonstrated that three cell

lines (FI9, F113, andF114) supportthereplication of tsB21,

thecomplementing effectwasexaminedmorequantitatively.

Plaque formation by tsB21at39°Cwasseen onthethree cell

lines containing ORF62 with titers as shown in Table 2.

Plaque formation was also seen on the 14SV-1

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ICP4-con-taining E5 cell line. Incontrast, tsB21 did notform plaques

TABLE 1. Complementation of tsB21 by transfection withcloned DNA fragmentsa

Plasmid Insert (PFU/ms) Fold

pBR325 4.7 x 102 1.0

pSG1 Contains ICP4gene 1.9 x 104 40.4

ofHSV-1

pVZVEcoE EcoRI Efragment 5.3 x 103 11.3

of VZV

pGl26 Contains ORF62 of 6.1 x 103 13.0

VzV

aCV-1cellsweretransfected with 0.5 ,ug of each ofthe indicated plasmids and 24 hlaterwereinfected with tsB21at39°Catamultiplicityof 2 PFUper

cell.Afterincubation for 20 hat39°C,cultureswereharvested and titerswere

determined.

LL1

0

> 0

N

-12.1 kb--rn

Z

L L11 E LL_

.-*. ---.9.2 kb

l 2 3 4 5 6 7 8 9

FIG. 2. Southernblotanalysis of cellularDNA.

Electrophoreti-cally separated EcoRI digests (10 ,ug) of cellular DNAs were transferred to nitrocellulose and probed with a 32P-labeled gel-purified EcqRI-E.Lane1contains15 pgofpVZVEcoEplus 10jigof VerocellDNA. Lanes 2(Vero),3(VN2),and 7(FIll)werenegative bythisanalysis.VZV sequencesarefound inlanes 4(FI6), 5 (FI7), 8 (FI13), and 9 (FI14). The autoradiograph shown is a 10-day exposure. Asingleband of>9.2kbin sizewas seenwithF19DNA (lane6) with alongerexposure.

efficiently on Veto cells under these nonpermissive

condi-tions.

Asimilarexperimentwascarried out withd120, amutant

of HSV-1 which containsa4.1-kb deletion inthe ICP4 gene (Table 2). At 37°C, d120 produced no plaques on Vero or

VN2cells butyielded manyplaqueson E5, F114, F113, and F19cells, with E5 cells providingthe most efficient support. Time course studies were conducted to demonstrate the relationship between the replication of the HSV-1 ICP4

mutants and time after infection. Cells were infected with

d120 at amultiplicity of2 PFU percell and maintained at

37°C. Ctiltures were harvested at the indicated times, and

titers weredetermined on E5 cells at 37°C (Fig. 3). The E5 cell lineefficiently complemented this virus, exhibiting fold increases(relativetoVN2cells) of2.6 x 104at24h and 2.6

x 105 at 48h, similarto what has been reported earlier (30). FoldincreasesforF114cellswere 458 at 24 h and 2.3 x 104 at48 h.Althoughthedegree of complementationwasless for the VZV-derivedcell lines than thatforE5

cells,

significant

TABLE 2. Titer of HSV-1 ICP4mutants on various celllinesa

Cellline tsB21titer (PFU/ml) d120titer (PFU/ml)

Vero 2.0 x 102 <101

E5 1.7 x 108 4.0 x 107

F114 1.0x 108 1.2 x 106

F113 9.8 x 107 4.0x 105

F19 3.6 x 107 2.7 x 104

VN2 NDb <lo,

a Each of the indicated cell lineswas seeded in duplicate into

60-mm-diameter dishesand infected withserialdilutions oftsB21 ord120.Following virusadsorptionfor 1 h, the cells were overlaid with maintenance medium

supplementedwith0.2%humanimmuneserumglobulin (6) and incubated for 7daysat39°Cfor tsB21 or at37°Cford120,after which the cells were fixed and stained with 0.1%crystalviolet.

bND. Not determined.

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imideperml, the only HSV proteins present in the infected cells are the structural proteins of the infecting virions,

- E5

suggesting

that a virion

protein

of HSV stimulates the

expression of VZV ORF62.

Western blots of cellscontainingORF62. Cell lysates from _0 F114 F114, F113, and F19 cells were examined by Western blot 6- Fl13 analyses and probed with antibody raised in rabbits against a

peptide synthesized according to the predicted C-terminal

/1_0

F19 amino acid sequence of ORF62. In the absence of infection,

the cell lines contained no detectable VZV polypeptides. However, infection with HSV-1 strain KOS (Fig. 5A) or tsB21(data not shown) led to production of proteins reactive with the ORF62 antibody. The predominant reactive moiety detected in the infected cells was 175 kDa in size. This protein was not seen in KOS-infected or uninfected VN2 cells butwas seenin lysates from VZV-infected HFF cells. When ORF62 antibody was preincubated with the ORF62 synthetic peptide (Fig. 5B), the 175-kDa protein was not soVN2

detected,

indicating

that the Western blotis

specific

forthe

geneproduct ofORF62. Wecall this product VZV "IE"175

because of its apparent molecular weight and functional homology to HSV immediate-early gene ICP4. As in the Northern hybridization analyses, we were unable to detect , | , ffi , VZVprotein in the absence of HSV infection, again

suggest-20 30 40 5 6 70 ing that a component of the HSV virion activates the 4nIRD ATFR INFECTION expression ofVZV "IE 175.

Mvvno Pr En111rr-%1I RvUM

FIG. 3. The growth of d120 in cell lines F19, F113, and F114, containingORF62, and E5 cells compared with its growth in VN2 cellsat37°Covertime.

complementation did occur. The VN2 cell line does not

contain VZV sequences and served as the nonpermissive

line. Complementation of tsB21overtime was also studied

with lines F19, F113, and F114. FI14 cells produced the

greatest degree of complementation, exhibiting fold

in-creases(relativetoVN2cells) of 137at24 h, 78at48h,and

292at 72h (datanot shown). These results indicate that the

VZV protein present in these infections is able to

comple-ment two different ICP4mutants of HSV-1.

Progeny viruses recovered from d120-infected E5, VN2, and F114 cells at 24 h were screened for the presence of recombinantprogeny. Undiluted culture materialwasplated

on Verocells at 37°C. Noplaques were observed.

Transcription in cells containing ORF62. Transcripts that

havebeenmappedtotheshortrepeatregionof VZVinclude

a 4.3-kb RNAcorresponding to ORF62 (26). We sought to detect a similar transcript in the transformed cell lines

containing ORF62. Total cellular RNA was isolated from

F114, F113, and F19 cell lines and Northern(RNA) hybrid-ization analyseswere performed by using32P-labeledpGI26

and pVZVEcoA as probes. VZV transcripts were not de-tected when 20,ugof RNAwere probedunderthese condi-tions.However,a4.3-kbtranscriptcould be demonstratedin F114cells following infectionwithtsB21at39°C (Fig. 4).The 4.3-kb transcript was present in increased amounts when infection withtsB21wascarriedoutin thepresenceof 50jig of cycloheximide per ml. VZV-specific RNA was neither detected in uninfected F114 cells treated with cyclohexi-mide, nor wasit seenin uninfected VerocellsorVerocells infected withtsB21.

Although tsB21 doesnotreplicate efficientlyin Vero cells at 39°C, immediate-early proteins are synthesized in these

cells(12).None oftheseimmediate-earlyproteinsis synthe-sized- in the presence of cycloheximide. Therefore, when tsB21 infectionoccurs inthepresenceof 50,ugof

cyclohex--0 C.,

a)

N

x

___ U

-4-CN Ce4

x m m

U

-4-'

LL 11 LL IL

cs

CN

n

4-2

0

I .

4.3

kb-1 2 3 4 5 6 7

FIG. 4. Northern blotanalysesof total cellular RNA. Eachlane contains 2 ,ug of cellular RNA. The blot was hybridized with

32P-labeled pVZVEcoA. Chx indicates that cycloheximide was

addedtothe culture. An18-hautoradiographicexposureisshown.

107

106

1o5

iL

o4

t 10

103

2

101

1oo

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LI 5LIn )-" J:

>- v -= L

I ii: - t:-. .. L.L t.> E > >

z.!

IC 'Id No

r Z Z

175kDa ... 4U

.0

I r

A

B

FIG. 5. Westernblot anrlysesof celllysates.The celllysatesinpanelsAand BwerereactedwithantibodytoasyntheticORF62peptide. The antibody usedforpanel Awaspreincubatedwith 100 p.gofsyntheticORF29peptideperml,and that used forpanelBwaspreincubated

with100,ug of synthetic ORF62 peptideperml. In bothpanels,thelanes contain thefollowingcelllysates:lane1,VZV-infected HFFcells; lane 2, mock-infected HFFs;lane3,KOS-infected F113cells;lane4,mock-infectedFI13cells;lanes5and6,KOS-infectedandmock-infected F114 cells, respectively; lanes 7 and 8, KOS-infected and mock-infected F19 cells, respectively; lanes 9 and 10, KOS-infected and

mock-infectedVN2cells,respectively.A2-dayexposureis shown. A 175-kDamoietyisseeninlysatesofKOS-infected F19 cellson a7-day

exposure.

DISCUSSION

In this report, we describe the complementation of two

different ICP4 mutants of HSV-1 by the product of VZV

ORF62. Plasmid pGI26, which contains this VZV gene, complements tsB21at39°C.Transformed celllines

contain-ingthisgenealso complementthis mutantat39°C.These cell lines also complement the HSV-1 ICP4 deletion mutant

d120, indicatingthat theproductofORF62cansubstitute for the HSVICP4 whennoICP4moietyispresent. Thedegree

of complementation exhibited by the cell lines was even greater than that seen following transfection of DNA

frag-ments (16) and provides further evidence for locating the VZVequivalent of ICP4to this regionof the VZVgenome.

Although a VZV-specific transcript was not detected in

uninfected cells containing ORF62 (FI14 cells), a 4.3-kb VZV transcript was seen following infection with tsB21. This transcript was also present in cells infected in the presence ofcycloheximide, indicating that an HSV virion protein activates the expression of this VZV gene.

More-over,we areabletodetect VZVproteinonlyintransformed

cells that are jnfected with HSV. This also suggests that a componentof the HSV virionactivatestheexpression of the incorporated VZV gene. Thus, since thisgene product can substitutefor theHSVimmediate-earlygeneICP4 and is 175

kDa, we have called it VZV "IE"175. Furthermore, al-thoughShirakiand Hyman (29) detected fourVZV immedi-ate-early proteins in their studies, our finding ofonly one

major protein moiety is commensurate with the fact that

pGI26 contains onlyone complete ORF.

Themost likely virion protein responsible for the

stimu-latoryeffect is the at-TIF (a-trans-inducing factor, Vmw65)

which has been localizedtothe HSVtegumentand which is knownto induce, in trans, the expression of HSV agenes,

including ICP4 (1, 4). Two upstream DNA elements are

thoughttobeinvolved in theresponseofthe HSV-1 ICP4to

o-TIF(3). One of these is the TAATGARATTC consensus

sequence (R = purine) which, if present, is sufficient for

transcriptional activation(3, 23, 37).A second is aGA-rich

motif, GCGGAAG, which isthought toactivate TAATGA

RATTC-like motifs with apoorconsensus(3).

We analyzed the DNA sequence upstream of VZV

ORF62, assigning the AUG of this ORF a value of +1.

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Scanning 1,000 bases upstream of the AUG, we noted a

TABLE 3. Regulatory elements upstream from thepredictedVZVORF62

Orien-Regulatoryelement Position" VZVsequence tationb

TATAbox -374to-386 TATATTATATAT +

CCATT box -396 to -400 CCAAT +

-468 to -472 CAAAT -521to-526 GCAATC

GAbox -514to-522 GCGCGAACG +

TAATGARATTC -586to -597 TTATGGGTTAT + -778to-787 TTATGGTTAT + Splbinding sites -568 to-573 GGCGGC

-630to -635 GGCGGG +

-647to-652 GGCGCG

-657to-662 GGCGGG +

-674to-679 GGCGCG

-684to-689 GGCGGG +

-701to-706 GGCGCG

-728to -733 GGCGCG

-755to-760 GGCGCG

-765to-770 GGCGGG +

"Relative

tothe

ORF62

AUG,which isassignedapositionof +1.

bTheorientation (+or-)isgiven withreference to the 5' to3'directionof

theassociated mRNA. R representsapurine.Sources of consensus elements

aregivenintext.

"I".

-L. I-Ir L.

.-. -.L. 6.-.

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number ofregulatory elements, includingsequences sharing

homology

to both TAATGARATTC and GA-box motifs

(Table 3).

AcandidateTATAbox is also shown inTable 3,

asare three

possible

CCAATboxes (15). Several putative

binding

sitesforSpl (22, 25) werealsofound.Although the

5'terminus of thetranscript associated withORF62 has not

yetbeen

mapped,

it is reasonabletoplace its locationwithin

this

region.

Our analysis therefore indicates that there are

regulatory

elements upstream ofORF62 that are similarto

those found upstream of the HSV ICP4 gene and which

could

respond

to a-TIF-like activities. Two genes adjacent

tothe HSV-1a-TIF(ORFAandORFB) modulate its activity

incotransfectionassays(24). Recent datasuggest that VZV

may encode a related protein, in that its ORF10 shares

significant predicted

amino acid homology with a-TIF (11,

24). Furthermore,

VZV ORF11 and ORF12 adjacent to

ORF10

share amino acid homology and identical ordering

along

thegenome with HSV ORFAandORFB(11, 24).

Therehave

recently

been otherreports inwhichcelllines

that

constitutively

contain

immediate-early

genes have been

utilized in studies ofregulation. Boom et al. (2) described

preparation

ofa ratcell linecontainingthemajor

immediate-early

gene of human cytomegalovirus. They, too, were

unable to find a

transcript corresponding

to this gene by

Northern

blotting.

However, treatment of their cells with

cycloheximide

for variousamountsof time ledto

expression

ofmRNAof the

expected

size. The

interpretation

ofBoom

et al.

(2)

was that

cycloheximide

treatment removes the

negative autoregulatory

effect ofthe humancytomegalovirus

major immediate-early protein

by preventing its synthesis.

The dataof

Stenberg

and Stinksi(33) alsoindicate that this

protein

has an

autoregulatory

role. In our studies, a

tran-script

was not seen in F114 cells in the presence of

cyclo-heximide; however,

infection ofthese cells in the presence

of

cycloheximide

led to an increase in the amount of

tran-script compared

with infection in theabsence ofdrug. One

explanation

for this is that VZV "IE"175 inhibits its own

transcription

aswell.

SmithandSchaffer

(30)

describetheuse of G418-resistant

cells

containing

the ICP4 gene of HSV-2 to

complement

d120.

Additionally,

E5 cells

(containing

theICP4ofHSV-1)

wereableto

complement host-range

mutantsof HSVtype2,

thus

showing

that ICP4

products

encoded

by

HSV-1 and

HSVtype 2are

functionally interchangeable during

produc-tive infection. In

comparison,

ourcell lines which express

VZV "IE"175 arealsoableto

complement

d120andtsB21.

It is of note that our three cell lines

complement

these

mutants to different

degrees

and that these results

parallel

the relative amounts of the 175-kDa

protein produced

on

infectionwithHSV. One

explanation

fortheseresults is that

the three lines differ in the percentage of cells that contain

ORF62 orinthe percentage of cells competent for infection

with HSV. An alternative

hypothesis

is that allcellscontain

ORF62 andareinfectioncompetent but their

ability

tomake

VZV

protein

differs.The latter

theory

would

imply

that VZV

"IE"175is

required

in

stoichiometric,

rather than

catalytic,

amountsfor

replication

to occur.

In summary, we offer evidence that the gene for VZV

"IE"175is

responsive

toavirion

protein

of

HSV-1,

presum-ably

a-TIF.

Sequence

elements

appropriate

to such an

interactionarepresent upstream of the gene. VZV sequence

data alsoindicate the presenceof

homologs

of HSV-1 a-TIF

(ORF10)

and its modulators

(ORF11

and

ORF12)

(11, 24).

This is in contrast to the situation for

pseudorabies virus,

another

a-herpesvirus;

its

major

immediate-early

gene is

a-TIF

responsive,

and the upstream sequence ofthis gene

contains elements of theTAATGARATTCmotif;however, pseudorabies virus itselfhas been reported to not have an

a-TIF homolog (5). We also provide evidence that VZV

"IE"175 inhibits its own transcription, an additional prop-erty that it shares with its HSVanalogICP4.

ACKNOWLEDGMENTS

WethankKenneth D. Croen for helpful suggestions and HollyA. Smith and WilliamC. Reinhold for technicalassistance.

P.R.K.wassupported byaPublicHealthService grantAl 18449 from the NationalInstitute ofAllergy and Infectious Diseases.

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