Mapping and expression of a human cytomegalovirus major viral protein.

0022-538X/84/100129-07$02.00/0

Copyright

© 1984, American

Society

forMicrobiology

Mapping and Expression of

a

Human

Cytomegalovirus Major Viral

Protein

MICHELLE GALL

DAVIS,'*

ENG-CHUNGMAR,1 YUAN-MINGWU,1t AND ENG-SHANG

HUANG1'2'3

DepartmentofMedicine,2Departmentof Microbiology andImmunology,3 and Cancer Research Center,1 University of North Carolina, Chapel Hill, North Carolina27514

Received 12 March 1984/Accepted 23 June 1984

Weconstructed a DNAfragmentmap of low-passageTownestraincytomegalovirus by analyzingcross-blot hybridization and hybridizationsofisolated recombinant clones. Theabundant latetranscriptswere located on this map by hybridization oflabeled total RNA ofvirus-infected cells to blottedDNA fragments. The most abundantlatetranscript, carriedbythe 11.7-kilobase EcoRIfragment(EcoRI-G), wasprecisely mapped. The EcoRI fragmentwas fragmentedand subcloned in a plasmid carryingsimian virus 40sequences (pSV-OH, constructedby Chi-Bom Chae, Department of

Biochemistry,

Universityof NorthCarolina, Chapel Hill).One resultingrecombinantplasmid,

pHD713SV2,

wastransferredtosimian virus 40-transformedmonkey kidney cells(COS-1) byDNAtransfection. Synthesisof acytomegalovirus-specific 67-kilodaltonproteinwasdetected in thesecells by reaction of blottedproteins withvirus-specificmonoclonalantibody.The 67-kilodaltonprotein isa major phosphorylated proteinfoundinvirions; it is not glycosylated. The location ofthegene for this 67-kilodalton protein is thereforeassigned to the center of the L-unique region of human cytomegalovirus, at 0.37 to 0.39map units.

Humancytomegalovirus (HCMV) isaherpesvirus associ-ated withclinical manifestations ranging from asymptomatic infection to congenital abnormality, mental retardation, in-trauterine death, or, inorgantransplant patients, interstitial pneumonia. This virus can infect at multiple sites, and its antigenic diversity allowssuperinfection of seropositive indi-viduals (9). Recently, it has been suggested that this virus has oncogenic potential comparable with those of other herpesviruses. Cytomegalovirus(CMV)wascapableof stim-ulating cellular macromolecular synthesis (19) and trans-forming mammalian cells in vitro (14).

Physical maps of the double-stranded DNA genome of some

laboratory

strains have been constructed(3, 18; R. L. LaFemina and G. S. Hayward, personal communication). In

addition,

recombinant DNA clones of these strains are available; this development will allow workers to define functional genes and compare specific portions of the genome from

'different

strains to study gene function and

expression.

We have isolated recombinant DNAclones of CMV restriction fragments derived from Towne strain, passage 36, andconstructed the DNAfragmentmap shown in Fig. 1. The restriction enzyme sitesare not identical to those mapped by LaFemina and Hayward (Lafemina and Hayward, personal communication) for a high-passage Townestrain.

In thiscommunication,wedescribeourstudy of abundant late transcripts. One of the most abundant late transcripts has been mapped to a small restriction

fragment,

and we have determined that this gene codes for one of the most abundant proteins in infected cells at the late stage of infection.

MATERIALS ANDMETHODS

Virus and viral DNA. HCMV Towne

strain,

passage 36,

was used for all

experiments.

Virus was grown in WI-38 * Correspondingauthor.

tPermanent address: Wuhan Institute of

Virology, Academy

Sinica, Wuhan,China.

humanfibroblasts, and virusDNA wasisolatedas

previous-ly described (8).

Enzymatic fragmentation of virus DNA andgel electropho-resis. Restriction enzymes were purchased from Bethesda Research Laboratories (BRL), and digestions were per-formedasrecommendedby the

supplier.

Agarose gelswere run aspreviously described (17).

Transferof DNA restrictionfragmentsingelsto nitrocellu-lose. DNA was transferred to nitrocellulose paper

by

the procedure by Southern (17). Gelsweresoakedon0.5NHCI solution for 15min and thenwere transferredto a solution containing0.5 MNaOH and1MNaClfor 15min todenature the DNA. Finally, gelswereneutralizedby soaking fortwo 15-min periods in a buffer solution containing 1 M Tris

hydrochloride

(pH 7.4) and 1.5 M NaCl. The DNA was transferred by blotting a transfer solution of6x SSC (lx SSC is 0.15MNaCl plus 0.015Msodiumcitrate) throughthe gels, nitrocellulose paper, and absorbentpaper.

Nick-translation of DNA. DNA waslabeledfor hybridiza-tionprobe by incorporation of

[a-32P]deoxyadenosine

mono-phosphate. Each nick-translation mixture contained 0.5 ,ug

ofDNA,25

,uCi

of

[a32P]-dATP

(ICNBiomedicals, Inc.),0.1

mM dCTP, 0.1 mM dGTP, 0.1 mM TTP, 100 mM Tris hydrochloride (pH 7.5),20 mMNaCl,5 mM

P-mercaptoeth-anol,

5mM

MgCl2,

2U ofPoll(BRL)and 0.01 ,ugofDNase (Worthington Biochemicals Corp.)per ml.After1h at12°C, reaction mixtures wereappliedtoSephadex G-100 columns eluted with10 mMTrishydrochloride buffer(pH8) contain-ing1 mM EDTA, and the excluded peakwaspooled.

Nucleic acid hybridizations. Hybridizations were done either in 6x SSC-0.1% sodium dodecyl sulfate

(SDS-1Ix

Denhardt solution at65°C for20 h or in the same solution containing 10%dextran sulfateand 35%formamide at

45°C

for 20 h. Probe DNA wasdenatured

by

boiling along

with competing nucleotides, including (per

milliliter)

10 ,ug of

polyadenylic

acid, 50 ,ug of calf thymus DNA, 50 ,ug of salmon testis DNA, and 50

p.g

ofyeastRNA; in

hybridiza-tionsinwhich bacterialDNAwasadheredto

filters,

10

p.g

of E. coliDNAper mlwasaddedas a

competitor.

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130 DAVIS ET AL.

tions of competing nucleotides are expressed as final

amounts in the hybridization solutions. All filters were

washed afterhybridization, first at roomtemperature for5 min in 2x SSC, and then three timesat 55°C for 20min in O.lx SSC with 0.1% SDS. X-ray exposures ofhybridized

filtersweresometimesenhancedbyaCronex Lightning-Plus

intensifying screenand exposureat -70°C.

Cloning of DNA fragments of HCMV. A collection of recombinantplasmids carrying HCMVDNAwasgenerated

by enzymatic recombinations of EcoRI-digested DNA of HCMV Towne strain, passage 36, and EcoRI-digested

pBR322 plasmid DNA. Ligation mixtures (20 jil)contained

A

B

0 b

C ...

0.5 jigof pBR322 plasmid DNA, 1 jigoffragmentedHCMV

DNA, 50 mMTris hydrochloride(pH 7.5), 10 mMNaCl, 10 mMdithiothreitol,50 jigofbovineserumalbuminperml,0.1

mM ATP, 25 mM MgCl2,and 1 U of T4 ligase (BRL). After incubation at 110C overnight, the mixture was used to transform E. coli LE392 to ampicillin resistance. Bacterial colonies were tested for hybridization to HCMV DNA by the procedure described by Grunstein and Hogness (6). Some fragments eluted from gels bya procedure described

by Maxam and Gilbert (12)were recombined similarly.

Subclonesof EcoRIrecombinantplasmidsweregenerated

by redigestion of the pBR322 recombinant plasmids and

0,0 0.7 02^ 0.3 0.4 0.5 0.6 0.7 0.8 0.9 .0

A 1.lA

A~~~p

" {VCK ...1ltJb'}UP'PLZXifflAlr:|LJ5*

C\ (MO1. CIULAR

cc.sE I- RAV,MY. N I L.L'NG;'Il;

IIt 4

D A

C 18.4

1) 1.2

E 15.9

14.7

11 .5

1 .9

8.7

K; 6.4

1. 5.8

1: 5.8

5.6

O 5.1

P34.7

4.2

Kt 4.1

1[ 3 .5

2.9

W- 2.0

X 1.7

1' 1.6

7 1 .5 Si 7 .5

L.z ~~4.2

1?DNA EcoRX

Fragmsent

A B

C

D

E

F

pill1) PLASM1I)

3

4 5 6 7

1(1 9

10

11

1(0i

13

1 4

15

16

17

18

1 9

2 0 21

22

2 3

1(1

1 0 1

34

31

22 I 7 6

s . 9

.6 4.9

3e6

FIG. 1. Mapof therecognition sites forEcoRIendonucleaseonthe DNAofCMVTownestrain, passage36.(A)Fractionallengthofthe DNAmolecule. (B) Recognitionsites for EcoRIendonuclease.A,Sites which differ between thismapand thesitesdetermined by LaFemina

andHayward (Lafemina andHayward,personalcommunication),andA,DNAfragments which encode abundant latetranscripts. (C)X-ray

exposureofend-labeled fragments of ADNA (a)andHCMV DNA (b)digested with EcoRI endonuclease. (D) Molecular length in kilobases of eachEcoRI fragment and the designation ofthe recombinant plasmid carrying the fragment. Plasmid pHD101 is a pBR322 recombinant

carrying HindIII-A of HCMV.Fragmentswhichdonotappear onthismaparederived from the L-S junction (fragmentsPand Q)orappearto

hybridize with fragmentsXand Zoncross-blot hybridization(fragmentYandfragments smallerthan 1.5 kb)but donotoccurin plasmid

pHD101.

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EXPRESSION OF CYTOMEGALOVIRUS PROTEIN 131

(A.)

pBR322 Hind m + SV40 Hind m

ligation deletion Avol-,Pvu

1.0kb R

)1D 1.7 kb

pSV-OH (4.84kb)

EcoRI

1.8 kb HqRI

mS

~~~~~~~~(C

H RNA

H

1.45 kb

(B.)

III

pHD7(pBR322-EcoRI-G)

j Bgl 11

11.3 kb 0.7 kb 2.9 kb +M13mp7

(7.2kb) ligation *BomHl

R 2.9 kb R

ATCCGGAATTC GAATTCCCCGATCT

M 13-713 (10.1kb)

I*

Eco RI (M13mp7)7.2 kb

R H + HR RNA

ltion 2.9 kb

2.15 kb

H.

ikb

XR /

l.1kb\ H

RNA isolation. Cells were lysed with 7 M guanidine hydrochloride and Sarkosyl and then layeredover 6 M CsCl solution and centrifuged to separate RNA and DNA, as described by Seeburg et al. (16). RNA was extracted from the pellets.

RNA gel electrophoresis. RNA was separated on 0.8% agarose gels containing 6% formaldehyde and 20 mM mor-pholinepropanesulfonic acid-acetic acid bufferat pH 7and 1 mM EDTA. After electrophoresis, RNA was blotted onto

a

b

i10..:

:*.

...

_

-A S'

do.

Om

c

d

e

f

_i.

6-

-

4

-

0

go~~

-_

pHD713 SVI

[image:3.612.68.277.75.389.2]

(8.7kb) pHD713 SV2(8.7 kb)

FIG. 2. Derivation of plasmids pHD713SV1 and pHD713SV2. (A) Plasmid pSV-OH was generated byrecombination of pBR322

( ~)andSV40(IZZ)DNAs.Thedirection of ori is thesame as

the early promoterof SV40. (B) Fragments resulting from BgIII

digestion ofplasmid pHD7wererecombined with M13mp7 replica-tiveform DNAdigested with BamHI. The EcoRI sites are 10bp

from the BamHI sites of the bacteriophage DNA.= l, HCMV DNA. (C) The 2.9-kb fragment resulting from EcoRI digestion of M13-713 DNA was recombined with EcoRI-digested pSV-OH

DNA.Recombinantplasmidsweredigested with Hinfltodetermine theorientation of theinserted CMV DNA (datanotshown). Enzyme sites indicatedare: R, EcoRI; D,HindIlI; K, KpnI;and H,Hinfl.

enzymatic recombination with other vectorDNAs (see the

legend toFig. 2for detaileddescription ofsubclones). The plasmid pSV-OH was isolated by C.-B. Chae of the Department of Biochemistry at the University of North CarolinaatChapelHill(unpublished data).Theplasmidwas

generated by enzymatic recombination ofHindlIl-digested simian virus 40(SV40) DNAand HindIll-digested pBR322 DNA. A deletion between the AvaI siteat 1,424 base pairs (bp) and the Pvull site at 2,065 bp on pBR322 was also introducedto generate a4.8-kilobase (kb) vector, including

SV40 sequences between the HindlIl site at 5,107 bp of SV40 and the origin ofSV40DNAreplication and between theKpnI site at230bpand theHindIII siteat 982bp(C.-B. Chae,personal communication). Adiagramof theplasmidis included in Fig. 2, indicating the direction of the SV40 sequencesandtheHinfl sitesofpBR322 whichwereusedto orient the CMVsequencesin therecombinants pHD713SV1 andpHD713SV2.

Otherrecombinantsweregenerated by enzymatic

recom-bination ofHindIII,BamHI,orBglII-digested HCMV DNA and plasmid pBR322 DNA or replicative form DNA of

[image:3.612.316.554.201.618.2]

bacteriophage M13mp7.

FIG. 3. Hybridization of 32P-labeled RNA isolated from cells infected with HCMV with nitrocellulose filters carrying HCMV

DNA. Cells were labeled with [32P]orthophosphate 60 h after infection, and RNAwasisolatedasdescribed in thetext. Nitrocellu-lose strips containing EcoRI fragments (lanes a and d), XbaI

fragments (lanes b and e), andHindIllfragments (lanescand f)were

hybridized with nick-translated HCMVDNA(lanesa toc)or32p labeledRNA(lanes dtof). Thefragments indicatedareEcoRI-G,

M, and LT (lanes a and d), Xba-B and LT (lanes b and e), and HindIII-Cand LT (lanescandf). The major fragments correspondto

themaplocation intheL-unique region indicated in Fig.1,and the LT fragments and EcoRI-Mcorrespondtotheterminal and junction regions ofcytomegalovirus DNA.

am

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132 DAVIS ET AL.

TABLE 1. Hybridization ofRNA versusplasmidDNA immobilized onfilters'

Plasmid RIfragment cpm

pHD7 G 3,825

pHD9 I 504

pHD14 N 1,400

pHD15 0 302

pHD20 T 748

pHD22 V 242

pHD13 M 2,291

a 2 ,ug of plasmid DNA was immobilized onnitrocellulose filters, and these werehybridized with 3.4 x 107cpmof RNA isolated from WI-38 cells 60 h after infection with HCMV. Duplicate samples were averaged, and the background radioactivity from filters carrying pBR322 DNA (168 cpm) was subtractedfrom each sample.

nitrocellulose in the same manner as described for DNA, with lOx SSC as atransfer solution.

Transfer of DNA to cells in tissue culture. Cellsgrownin 5-cm plastic wells were treated with CMV DNA by the technique described by Graham and Van der Eb (5) as modified by Stowe and Wilkie (20). The DNA-calcium phosphateprecipitates containingS ,ugofplasmidDNAand 10 ,ugof calf thymus DNA were added to subconfluentcell monolayers,and the treatedcellswereincubatedat36°Cina CO2 incubator with occasional agitation for 40 min. Ten milliliters of minimal essential medium (MEM) containing 10% fetal calfserum was added, and after4 h the medium wasreplaced with 2mlof15%

dimethyl

sulfoxide in MEM. After 4 min at room temperature,

dimethyl

sulfoxide was removed, andthecellswerewashedwith MEM.

Finally,

10

a b c d e f 9 h i k

*041(

,-WwIll,0w

probe: _rDNA

406o

HCMV

1.9kb

pHD7

FIG. 4. Hybridization of 32P-labeled DNA with RNA

immobi-lized onfilters. RNAwas separated onagarose-formaldehyde gels, blottedontonitrocellulosefilters, and hybridizedwith

nick-translat-edDNA.RNAwasisolatedfrom uninfectedcells(lanesa, e,andi),

cells infected with HCMV 60h previously(lanes b, f, and j), cells infected with HCMV and treated with phosphonoacetic acid (100 p.g/ml)for10h(lanesc, g,andk), and cellsinfectedwith HCMVand

treated with cycloheximide (50 p.g/ml) for 4 h (lanes d, h, and 1). Nick-translated DNAprobesare:rDNA, plasmidpSF2124carrying

DNAfrom theseaurchin Arbaciapunctulata specificfor 18SRNA (M. Davis, unpublished results); HCMV, total virus DNA;pHD7, plasmid pBR322 carrying EcoRI-G of HCMV. The molecular weights indicatedwere estimated from the size of18S RNA(1.869 kb)and other gels in which pBR322 DNAfragmentswereappliedto

the gels.

ml ofMEM

containing

10%fetalcalf serum wasadded, and the cells wereincubatedfor 24 h and then harvested.

Detection of HCMV-specific antigens. Proteins from the treated cells were

analyzed by

SDS-polyacrylamide

gel

electrophoresis by

the method ofLaemmli(10),as

previous-ly

described

(11).

Proteinswereelectroblottedonto nitrocel-lulose

sheets,

as described

by

Towbin et al. (21). The identification of

HCMV-specific antigens

was

accomplished

by

exposing the nitrocellulose sheets to monoclonal

anti-body

(the detailsof theproductionofmonoclonalantibodies to HCMV Towne virion

proteins

will be published else-where)and then stainingthemwithhorseradish peroxidase-conjugated immunoglobulin G (IgG), 4-chloronapthol, and

H202,

as described by Hawkes et al. (7). A blue color reactionindicated the presence ofantigen.

Immunoprecipitationof CMVproteins. Mock-or HCMV-infectedcellswerelabeledfor 20 hat72 hpostinfection with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate as described by Mar et al. (11). Cells were harvested

by

scraping, washed three times withTris-buffered saline, and solubilized with RIPA buffer (0.05 M Tris

hydrochloride,

0.15 M NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100,0.1 mMphenylmethyl sulfonyl fluoride) for30 min on ice. The lysate was sonified and centrifuged in a microcentrifugefor15 min. One-half milliliter of supernatant wasmixed with 10 pu1 of ascites fluid from hybridoma clone 185E, 132-5, H-644, orH176 and with 100 p.l ofanti-mouse IgG (whole molecule)-agarose (Sigma Chemical Co.) and gently agitated at 4°C overnight. The agarose beads were collected bycentrifugation, washed five times with 1 ml of RIPAbuffer, and suspended in 50 p.1 ofsamplebuffer(0.37 M Tris hydrochloride [pH 6.8], 15% glycerol, 5% ,3-mercap-toethanol, 1.5% SDS) for polyacrylamide gel electrophore-sis. Afterheating at90°C for5 min, the sample wascooled, thebeads werepelleted, and the supernatant proteins were loaded onto a9% polyacrylamide gel. Detection of [3H]glu-cosamineand35S-labeled proteinswasdoneby

fluorography

(1),anddetection of32P-labeledproteins wasdonebydirect exposure of gels to X-ray film, enhanced by a Cronex Lightning-Plus intensifying screen.

RESULTS

Physical map of HCMV Towne strain. Each recombinant plasmid was nick translated and hybridized to blotted re-striction

fragments

of total viral DNA. Theresults ofthese experiments were compared with data obtained by cross-blothybridization. Inaddition, restrictionenzyme sites were mapped on the recombinant plasmids. Physical maps of strains Davis (3), AD169 (18), and Towne (LaFemina and Hayward, personal communication) are similar to the ones showninFig. 1, but the regions at eachendand near the L-S junction vary. Thesepointsofvariability betweenthe maps of high- and low-passage Towne strain are indicated by asterisks.

Mapping regionsoftranscriptiononthegenomeofHCMV. Hybridizationof32P-labeled RNA isolated from cellsatany time between 14 and 96 h after infection with filters contain-ing restriction enzyme fragments ofHCMV DNA yields a complex pattern (Fig. 3), indicating that many regions are beingtranscribed (22). Wefocused onthe sites ofabundant transcription at the late stage of infection as shown by the intensity of the hybridization of labeled RNA withblotted DNA fragments (Fig. 3 and Table 1) and by the reverse method, hybridization of nick-translated DNA fragments with RNA immobilized on nitrocellulose filters

(Fig.

4). In Fig. 4, the hybridized filters were

developed

after short

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A a b c d e a b c d e

_

9

/

:

FIG. 5. Physical map of plasmid pHD7. Anagarosegel(left) carrying restriction enzymefragmentsofplasmid pHD7 was blotted onto nitrocellulose paper whichwashybridized with 32P-labeledRNAisolated from cells60 hafter infection with HCMV(right). Lanes: a,XDNA digested withHindlll;b,undigestedplasmidpHD7; c, pHD7digestedwithEcoRI andBamHI; d, pHD7digestedwithEcoRIandBglII;ande, pHD7digestedwithEcoRl,BamHI,andBgIll. (B)AmapofplasmidpHD7indicatingtheorientation ofpBR322 (4.4kb) and sites for EcoRi (R),BamHI(B),BglII (G), and Hinfl (H). The orientation shown is the orientation offragmentEcoRI-Gonthemap shown inFig. 1, and the transcription of1.9kb RNA is from lefttoright.TheHinflsites have beenmappedby DNA sequencing experiments (data not shown). Only the terminalHinflsitesareindicatedonthe 2.9-kbBglII fragment.

exposure, to show only the most intense

labeling.

Two distinct regions of

transcription

located

by

these experi-ments are indicated below theEcoRI restriction map (Fig. 1), one of these near the L-Sjunction and the otherat the middleof theL-unique region. Much of the complexpattern ofhybridization ofRNAwithEcoRI

digestion fragments

can be attributed to the

multiple

L-S

junction

and

L-repeat

fragments

which are distributed

throughout

the size range (LaFemina and

Hayward, personal

communication)

and which hybridize to an abundant 1.1-kb RNA

(Fig.

4 and Table 1). This transcription region is represented at least twicein every virusmolecule,attheL-Sjunctionandatthe L terminus. At longer exposure, probably all restriction fragments of HCMV hybridizeswith32P-labeledRNA isolat-ed fromcells late ininfection.

A comparison of the abundance of these two major transcripts with others was performed by

hybridizing

32p_ labeledRNAinsolutionwith filterscontainingrecombinant plasmidDNA(Table 1).

The results are comparable with those

previously

deter-minedfortotal viralDNAblots, although neither experiment is correctedtomolarity byestimating the sizes of theRNA. Fragment G carried by plasmid pHD7 was labeled 15-fold more intensely than fragments V or 0, and fragment M, which carries part ofthe L-repeat DNA, wasninefoldmore radioactive thanfragment V. Asdemonstrated in Fig. 4, the RNAhybridizing to fragment G is a latetranscript of 1.9 kb. ThisRNA was not present in cells treated with cyclohexi-mide orphosphonoacetic acid after virus infection.

Isolation of one region of transcription. The most intensely labeled fragment, EcoRI-G carried by plasmid pHD7, was fragmented by enzymeBglIIinto fourportions,oneof which hybridizedtoRNA isolated frominfected cells(Fig. 5). This 2.9-kbBglIIfragmentis theonlytranscribedregionwehave detected in theoriginal 11.7-kb EcoRIfragment.This obser-vationwasconfirmedbyelectronmicroscopeobservation of R-loops, which showasingleuninterruptedregion of hybrid-ization (L. Loh and E.-S. Huang, unpublished data). The 2.9-kbBglII fragmentwassubcloned into the BamHI site of bacteriophage M13mp7, and the fragment which was

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134 DAVIS ET AL.

leasedafterEcoRI digestionof thereplicative-formDNA of the recombinant M13-713 was recombined into the EcoRI site of plasmid pSV-OH (Fig. 2).

Plasmidsresultingfrom the recombination between M13-713 and plasmid pSV-OH were divided into two groups, based on digestion patterns with enzyme Hinfl. Plasmid pHD713SV1 carries the inserted region in the orientation opposite to that inplasmid pHD713SV2 (Fig. 2).

Expression of the 67K protein in tissue culture cells. Cul-tures of HEL cells and COS-1 cells were treated with plasmidDNA asdescribed above,andproteinwasextracted and applied toacrylamide gels asdescribed. Electroblotted protein was reacted with monoclonal antibody prepared in thislaboratory specificfor a 67,000-molecular-weight (67K) protein of HCMV. A photograph of the stained nitrocellu-losepaper is shown inFig.6. The 67K protein wasproduced in COS-1 cells carrying pHD713SV2 DNA but not in cells carryingpHD713SV1 DNA. Theorientation ofthe transcrip-tion ofthe 67K proteinsequence in plasmid pHD713SV2is downstream from the major early promoter of SV40. In plasmid pHD713SV1, this orientation is reversed.

Characterization of the 67Kproteinininfected cells. WI-38 cells infected with HCMV were labeled with [3H]glucos-amine,

[35S]methionine,

or

[32P]orthophosphate

72 h after infection. Afterextraction of totalprotein, the 67K protein was immunoprecipitated with monoclonalantibody, andthe protein was analyzed by gel electrophoresis. The gel was exposed to X-ray film

(Fig.

7). The 67K

protein

was not labeled by

[3H]glucosamine,

but it was labeled by 32P and 35s.

DISCUSSION

The 67Kproteinis astructural protein of HCMV,infact, oneofthemajor proteins foundinisolated virions

(Fig.

6 and

cellsweretreated with theplasmid DNAindicatedorinfected with HCMV. Protein was extractedfrom the cultures and from isolated virions, isolatedas previouslydescribed (8), appliedtoacrylamide gels, and separated by electrophoresis. Electroblotted protein was reacted with monoclonal antibody and stained with horseradish

f5fel..,

1 2

'200K

15 0K " "

n.'. .'f

68K.

5x

3

32_ID /_;riSK_?

4 5 6

67K |..

inls

FIG. 7. Immunoprecipitationofpolypeptides ofHCMV-infected cells labeled with [35S]methionine, [3H]glucosamine, or [32P]ortho-phosphate byamonoclonalantibody. Theimmunoprecipitateswere subjectedtoSDS-polyacrglamidegelelectrophoresisasdescribed in the text. Lanes 1, 6, and 9 contain host cell proteins labeled with

[35S]methionine, [32P]orthophosphate, and [3H]glucosamine, re-spectively. Lanes 2, 5, and 8 containinfectedcellproteins labeled with [35S]-methionine, [32P]orthophosphate, and [3H]glucosamine, respectively.Lanes 3, 4, and 7 containlabeledpolypeptides precip-itated from HCMV-infected cellextracts by monoclonalantibody. No detectable [3H]glucosamine-labeled protein was precipitated (lane 7), althougha 67Kprotein was precipitatedfrom 35S-and 32p-labeled cultures.

7). The monoclonal antibody which reacts with the 67K protein does not neutralize virusinfectivity, indicating that the proteinmay be internal. Itprobably corresponds tothe 66K phosphorylated protein described by Gibson (4), a tegument protein which interfaces thecapsid and the mem-brane of the virus. A 64Kglycoprotein recently described by Clarket al. (2) is unrelatedto the 67Kphosphoprotein(Fig. 7). The monoclonal antibody preparation which precipitates the 67K protein fails to precipitate a labeled protein from infected cell proteins labeled with [3H]glucosamine. The gene for the 64K glycoprotein of Clark et al. is carried by fragment EcoRI-A (H. Pande, S. Baak, J. A. Zaia, B. R. Clark, J. E. Shively,and A. D. Riggs, Eighth International Herpesvirus Workshop, Oxford, abstr. no. 272, 1983).

The RNA transcripts that we describe have also been identifiedby Wathen and Stinski (22). We do not detect the larger RNAs that they describe, but the smaller species at 1.9 and 1.2 kb correspond to the map locations that we report for 1.9- and 1.1-kb RNA.

The intense transcription of L-repeat regions was also found in cellsinfected with strain Ad169 of CMV (13). The 1.9-kbtranscript we describe probably corresponds to RNA

peroxidase-conjugated IgG, 4-chloronaphthol, and H202. Only COS-1 cellscarrying DNAofplasmid pHD713SV2had detectable amountsofa67Kprotein.

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hybridizingtoEcoRI-IofAd169, althoughit isnot character-ized asthe most abundant transcriptfrom analysis of cyto-plasmic, polyadenylated RNA. McDonough and Spector (13) describe straindifferences in the pattern oftranscription in CMV-infectedcells.Spectoretal.(18)also havemappeda 3-kbBglII fragment (BglII-X) inEcoRI-I, flankedbya 0.78-kbfragment

(BglII-f).

The orientation of thesefragmentshas been determined by DNA sequencingand

cross-hybridiza-tion ofinternalfragments in this laboratory (Fig. 5). The L segment ofHCMV in maps of Ad169 is inverted relative to theorientationillustrated inFig. 1.Itappears that theregion near0.4map units is thesamebetween thesestrains; indeed, it is likely that most of the genomes of cytomegalovirus strainsare

genetically

colinear

(23).

Variationsareclustered attherepeattermini, andthese are

probably responsible

for nonhomologous sequences betweenstrains (15).

We have not determined whether the 2.9-kb

BglII

frag-mentcarriesafunctional promoter,sincecellsexpressedthe 67K

protein only

when transfected with recombinant

plas-midDNAofpHD713SV2whichcarriesHCMV DNA down-stream from an

early

promoter of

SV40,

and

only

COS-1 cells expressed the

protein

at adetectable level.

Expression

of the 67K

protein

was not detected in human

embryonic

lung cells transfected with either

pHD713SV1

or

pHD713SV2 DNAs. If a viral promoter is located on the BglII fragment, it does not promote the

expression

of the 67K

protein

when dissected from the total HCMV viral DNA.

Evidently,

an

early

orimmediate

early

viral function is required for

expression

of the 67K

protein.

We can

investigate this

requirement by

measuring protein

expres-sion in cells after transfer of

pHD713SV1

DNA in combina-tion with other recombinant

plasmids

orafter

microinjection

of HCMV macromolecules.

We identified a

putative

promoter on the

BglII

fragment

byDNAsequence

analysis

ofthe DNAinsertion ofM13-713 bacteriophage. The 5'end ofthe RNA islocated300

bp

from one

BglII site,

andno

intervening

sequencesweredetected. The DNA sequence 5' to the site of RNA initiation is abruptly different from the

surrounding

sequence. Five hundred

base pairs

fromtheinitiation

site,

theG+Ccontent

of the DNA is 66%. In contrast, the 230

bp

before the initiation site contain 29% G+C. The entire

coding region

including 700 bp downstream is 50%

G+C,

and the most distant sequence that has been

determined,

700 to 900

bp

after the

polyadenylation

signal,

is 66% G+C. A definite gene structureis evidentfrom this

analysis

(M. G.Davisand E.-S. Huang,

manuscript

in

preparation).

Theentiresequenceconsists of

surrounding

viralDNAof

high

G+C content,

abruptly

interrupted by

an extended A+T-rich

region,

followedby an informationsequence that contains maximum nucleotide

variability.

We

plan

similar analyses ofother

coding regions

of human

cytomegalovirus

todeterminethefunction oftheseregions,their

distribution,

and their

controlling

elements.

Analysis

of similar

coding

regions of other

herpesviruses,

identified

by

cross-hybridiza-tion oftheCMV

coding region

and restriction

fragments

of herpesvirus DNA, may indicate similar functions in other membersofthe virus group.

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