Proc. Natl. Acad. Sci. USAVol. 81, pp. 4965-4969, August 1984Microbiology

Cloning and physical mapping of a gene fragment coding for a64-kilodalton major late antigen of human cytomegalovirus*

(recombinant DNA/mixed-sequence oligonucleotide/Southern blots)

HEMA PANDEt, STEVEN W. BAAKt, ARTHUR D. RIGGS§, BRIAN R. CLARKt, JOHN E. SHIVELYt,AND JOHN A. ZAIAtDivisions of tImmunology and §Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010; and tDivision of Pediatrics, City of HopeNational Medical Center, Duarte, CA 91010

Communicated by Rachmiel Levine, March 16, 1984

ABSTRACT We have isolated a clone containing a genefragment coding for a 64-kilodalton glycoprotein that is themajor late antigen of human cytomegalovirus (HCMV). Basedupon the amino acid sequence of a tryptic peptide of this glyco-protein (HCMVgp64), two sets of mixed-sequence probes, oneconsisting of a mixture of 16 heptadecadeoxyribonucleotidesand the other a mixture of 32 icosadeoxyribonucleotides, weresynthesized. A subgenomic library of HCMV (Towne strain)DNA was constructed in plasmid pBR327 and transformantswere screened with 32P-labeled aliquots of these synthetic oli-godeoxyribonucleotide probes. Two clones among 15,000 gavestrong positive signals. Plasmid DNA was isolated from thepositive clones and characterized by restriction mapping andSouthern blot analysis using both probes. The plasmid DNAcontained a 2.3-kilobase insert, which yielded an 800-base-pairand a 1500-base-pair fragment after Sau3A digestion. Only the800-base-pair fragment hybridized to the mixed probes, andDNA sequence analysis revealed that it contains nucleotide se-quences compatible with amino acid sequences of tryptic pep-tides of HCMVgp64. Restriction mapping studies of HCMVDNA using this 32P-labeled 800-base-pair cloned DNA have al-lowed us to locate this gene fragment in the long unique regionof HCMV (Towne strain) genome at -0.5-0.51 map unit.

Human cytomegalovirus (HCMV), a member of the herpes-virus group, is associated with a wide spectrum of clinicalsyndromes, including congenital birth defects, mononucleo-sis, and interstitial pneumonia in immunosuppressed individ-uals (1-3). In addition, HCMV has an oncogenic potentialand a possible association with certain types of malignancyincluding Kaposi sarcoma (4-6).The HCMV genome is a linear double-stranded molecule

with an approximate size of 240 kilobase pairs (kb) (7). TheDNA consists of a long and a short region of unique nucleo-tide sequences that are bounded by inverted repeat regions(8, 9). HCMV genome specifies >50 unique infected-cellpolypeptides with molecular masses ranging from 200 to 10kDa (10-12). Of these, a major structural glycoprotein hav-ing a molecular mass of 64-66 kDa (HCMVgp64) is presentin high abundance during the late stages ofHCMV infection(13-17) and is an important antigenic component in serologictests for HCMV. Antibody to HCMVgp64 appears after nat-ural infection in both adults and children (15, 18). Monoclo-nal antibody specific for this viral protein neutralizes virusinfectivity and binds to the plasma membrane of infectedcells (14).To further understanding of the precise role of

HCMVgp64 in natural HCMV infection, we have attemptedto clone the gene coding for this structural protein. Clark etal. have recently purified this polypeptide from the virions

plus dense bodies (HCMV+db) of HCMV (19). The presentstudy describes the cloning of a gene fragment coding forHCMVgp64 by means of oligonucleotide probing. Sincenone of the structural polypeptides ofHCMV were previous-ly mapped, a significant aspect of this report is the physicalmapping of the coding sequences of HCMVgp64.

MATERIALS AND METHODSGrowth of HCMV. HCMV (Towne strain) was obtained

from S. Starr (Philadelphia, PA) and grown in human fore-skin fibroblasts. Growth media consisted of Dulbecco'smodified Eagle medium, supplemented with 10% fetal bo-vine serum. Cells were maintained in a 10% C02/90o airatmosphere at 36°C. Fibroblast monolayer cultures weregrown in 485-cm2 roller bottles and inoculated with cell-freevirus by using a multiplicity of infection of 0.1-0.2. Extracel-lular virus was harvested and HCMV+db were prepared asdescribed (19).

Isolation, Trypsin Digestion, and Microsequence Analysis ofHCMVgp64. HCMVgp64 was purified from HCMV+db byreverse-phase HPLC (19). Trypsin digestion and separationof tryptic peptides has been reported (19). Amino acid analy-ses of tryptic peptides were performed on a Beckman 121MB amino acid analyzer according to the procedures de-scribed earlier (20). NH2-terminal sequence analysis of thetryptic peptides was performed using microsequencing tech-niques by automated Edman degradation on a modifiedBeckman 890C sequencer by the method of Shively (21).

Synthesis of Mixed Oligodeoxynucleotides. Mixed oligonu-cleotides 17 bases and 20 bases long were synthesized by thesolid-phase phosphotriester approach (22) using protecteddinucleotides. Oligonucleotides were purified by HPLC on areverse-phase column (Waters Associates ,uBondapak C18).Labeling of the purified oligodeoxynucleotides at the 5' endwas performed by using T4 polynucleotide kinase and [y-32P]ATP. The radioactively labeled oligonucleotides werepurified on Whatman DE-52 DEAE-cellulose columns (23).After filtration (0.2-,um-pore Nalgene filters), the probeswere used directly for hybridization at a concentration of 0.2ng/ml per species of probe (about 107 total cpm).

Construction of Recombinant Plasmids. HCMV DNA wasisolated according to the methods of Demarchi (24) andStinski et al. (25) with the modification as described. Theviral DNA was released from purified virions by treatmentwith 2% sodium lauryl sarkosinate that was pretreated with150 ,ug of proteinase K per ml. After incubation at room tem-perature for 30 min the mixture was extracted with equalvolumes of phenol and chloroform/isoamyl alcohol (24:1) bygentle shaking on a platform shaker. The DNA was precip-

Abbreviations: HCMV, human cytomegalovirus; db, dense bodies;kb, kilobase pair(s); bp, base pair(s).*A preliminary report of this research was presented at the EighthInternational Herpesvirus Workshop, Oxford, England, 1983.

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itated from the aqueous layer with 95% ethanol. PlasmidpBR327 was used as a cloning vector (26). The subgenomicfragments of HCMV DNA generated by digestion with re-striction endonuclease Sau3A were ligated with BamHI-cutplasmid pBR327 using T4 ligase. The recombinant plasmidwas used to transform Escherichia coli strain LSI, a lac+derivative of RR1 (pro, leu, thi, rpsL20, hsdR, hsdM) (27) bythe procedure of Hanahan as described in ref. 28. Cloneswere selected on LB agar plates containing 20 pzg of ampicil-lin per ml.

Screening of HCMV Library. Recombinant bacterialclones were screened on Whatman 541 filter papers, ampli-fied with chloramphenicol (250 ,g/ml), and prepared for hy-bridization as described by Gergen et al. (29). The filterswere prehybridized in 6x concentrated SET buffer (SETbuffer = 0.15 M NaCl/0.001 M EDTA/0.015 M Tris HCI,pH 7.5) containing 100 ,g of sonicated salmon sperm DNAper ml and 0.5% Nonidet P-40. Hybridization to the probewas performed at room temperature in the same buffer. Fil-ters were washed first with 0.9 M NaCl/0.09 M sodium cit-rate, pH 7.2, at room temperature. Three high-stringencywashes were done at 43°C in the same buffer and then thefilters were exposed to x-ray film.

Hybridization of Probes to Plasmid DNA. Plasmid DNAwas prepared according to Ish-Horowicz and Burke (30).The 32P-labeled probes were hybridized to restriction en-zyme-digested plasmid DNA on dried agarose gel at roomtemperature for 16-18 hr. The buffers used for hybridizationand washes were the same as those used for colony hybrid-ization.DNA Sequence Analysis. DNA restriction fragments were

cloned into phage cloning vector M13 mp8 and single-strand-ed phage DNA was isolated as described by Messing andVieira (31). Nucleotide sequence analysis was performed bythe method of Sanger et al. (32) by using synthetic oligonu-cleotide primers.

Southern Blot Analysis. HCMV (Towne strain) DNA wasdigested to completion by using restriction enzymes EcoRI,BamHI, HindIll, and Xba I and by double digestion usingBamHI and HindIl. The restriction digests were electro-phoresed on agarose gels, stained with ethidium bromide,and blotted onto nitrocellulose (33). The cloned HCMV in-sert [800 base pairs (bp)] was nick-translated with [a-32p]-dCTP (800 Ci/mmol; 1 Ci = 37 GBq) using a nick-translationkit (Amersham). Hybridization was conducted in a mixturecontaining 0.9 M NaCl/0.09 M sodium citrate, 1.0 mMEDTA, 5x concentrated Denhardt's solution, 0.5% Na-DodSO4, and denatured salmon sperm DNA at 100 ug/ml at68°C for 16-18 hr. Filters were then washed with 15 mMNaClI/1.5 mM sodium citrate/0.5% NaDodSO4 at 68°C for 2hr (34), dried, and exposed to x-ray film.

RESULTSOligodeoxyribonucleotides. Overlapping synthetic oligonu-

cleotides were used as specific hybridization probes. Thenucleotide sequences of these probes were based on the par-

Anino acid sequence

Possible Codons

tial amino acid sequence of HCMVgp64. A heptapeptide se-quence derived from a tryptic fragment of HCMVgp64 wasselected for the design of two sets of oligonucleotide mix-tures containing all of the possible coding sequences (Fig. 1).Both probes were synthesized in one main synthesis. Thefirst set (designated as HCMV probe 1) was derived from thehexapeptide sequence Gln-Glu-Phe-Phe-Trp-Asp, and wassynthesized as a mixture of 16 heptadecanucleotides. At thecompletion of the synthesis ofHCMV probe 1, the resin wassplit and half of it was used for the synthesis of the secondprobe. The second probe (HCMV probe 2) was based on aheptapeptide sequence derived from the above hexapeptidebut containing an additional tyrosine at the NH2 terminus.This second set of oligonucleotides was synthesized as amixture of 32 icosanucleotides and involved two additionalcouplings to the second half of the resin.

Isolation of a Bacterial Clone Containing HCMVgp64 GeneSequences. The subgenomic fragments of HCMV DNA gen-erated by digestion with restriction endonuclease Sau3A(0.3-3 kb) were inserted in the BamHI site of the plasmidpBR327 by the standard DNA ligation method. The recombi-nant plasmid was used to transform E. coli strain LSI. Apermanent collection of transformed colonies was stored onnitrocellulose filters. Of the 15,000 transformants screenedby hybridization to 32P-labeled HCMV probe 1, two stronglyhybridizing colonies were identified. When HCMV probe 2was used for screening, positive hybridization was observedto the same two colonies. The selection procedure includedhybridization with probes at increasing temperatures inwhich the most stringent conditions approximate the predict-ed dissociation temperature (35). Plasmid DNA was pre-pared and inserts were analyzed by restriction enzyme map-ping. The Pst I-digested recombinant plasmid DNA wascompared to Pst I-digested plasmid pBR327 DNA by elec-trophoresis on agarose gel, and this showed an insert of =2.3kb. When the recombinant plasmid DNA was digested withrestriction enzyme Sau3A and compared with Sau3A-digest-ed plasmid pBR327 DNA, two additional fragments, an 800-bp and a 1500-bp fragment, were observed for both positivecolonies. Of these two fragments, only the 800-bp fragmenthybridized to the 32P-labeled synthetic probes on nitrocellu-lose filters by Southern hybridization method.

Nucleotide Sequence Analysis. The nucleotide sequenceanalysis of the 800-bp insert was obtained by the dideoxychain termination method (32). Subfragments of the 800-bpinsert were generated by using restriction endonucleasesHpa II and Sma I and subjected to nucleotide sequence anal-ysis after subcloning into bacteriophage M13 mp8 and mp9,respectively.

Fig. 2 shows the combined nucleotide sequence and thededuced amino acid sequences of the most relevant portionof the cloned insert. Clark et al. have described the trypticmapping of HCMVgp64 (19). Approximately 3 nmol ofHPLC-purified HCMVgp64 was used for these studies andthe recovery of tryptic peptides was >90%. The HPLC-puri-fied tryptic peptides were arbitrarily numbered from T-1 toT-14 and subjected to amino acid composition analysis. On

Tyr - Gln - Glu - Phe - Phe - Trp - Asp

5' UAuc CAA GAG UUu UUu UGG GAu 31'

HCMV probe 1

HCMV probe 2

5' CAA GAA TTc TTc TGG GA 3 '

5' TAT CAA GAA TTT TTT TGG GA 3'c CG GA TC TC TG GA 3

FIG. 1. Oligonucleotide probes used in the isolation and characterization of a genomic clone containing the gene fragment coding forHCMVgp64.

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Asn Leu Val Pro Met Val Ala Thr Val Gln Gly Gln AsnAAC CTG GTG CCC ATG GTG GCT ACG GTT CAG GGT CAG MT

Leu Lys I Tyr GIn Glu Phe PheCTG MG TAC CAG GAG TTC TTC

Probe - sequence

T-14Trp Asp Ala Asn Asp IleTGG GAC GCC AAC GAC ATC

T-12Ple Phe Al a Gl u Leu Gl u GlyATC TTC GCC GAA TTG GAA GGC

Val Trp Gl nGTA TGG CAG

Pro Ala Ala Gin Pro LysCCC GCT GCG CAA CCC MA

FIG. 2. Nucleotide sequence and the deduced amino acid sequences of a gene fragment coding for HCMVgp64. The nucleotide sequences

that show perfect match with the probe sequence are underlined. The regions where the predicted amino acid sequences are completelyidentical to the amino acid sequences of the two tryptic peptides are indicated by bracketed lines.

the basis of amino acid composition, we selected two trypticpeptides, T-12 and T-14, having retention times of 55.9 and59.4 min, respectively, and determined their amino acid se-quences. These sequences were then compared with theamino acid sequences predicted from the nucleotide se-quences of the cloned DNA. The deduced amino acid se-quences revealed total agreement with the sequences oftryptic peptides at the positions shown in Fig. 2.Mapping of the Gene Coding for HCMVgp64. The 800-bp

HCMV insert was gel purified, nick-translated, and hybrid-ized to HCMV DNA digested with restriction endonucleasesEcoRI, BamHI, Xba I, HindIII, and a BamHI/HindIII dou-ble digestion. Fig. 3A is an ethidium bromide-stained gelshowing the pattern of fragments generated after digestion ofHCMV DNA with EcoRI, BamHI, Xba I, and HindIII. Themolecular masses of the restriction fragments are similar tothose obtained by LaFemina and Hayward (R. LaFeminaand G. Hayward, personal communication). The HCMV re-striction fragments that showed hybridization to the labeledprobe were characterized and are shown in Fig. 3B. Theslowest moving EcoRI fragment of HCMV has a size of 23kb in our gels and hybridized strongly to the cloned frag-ment. In addition, strong hybridization was observed to a6.15-kb BamHI fragment and a large fragment (-30 kb insize) of the HCMV-Xba I digest. The HCMV DNA digestedwith HindIII and a BamHI/HindIII double digest (data notshown) gave a small fragment of -1 kb in size, whichshowed hybridization to the same 800-bp probe. We haveused the results of these Southern blots to locate the clonedgene fragment coding for HCMVgp64 on the HCMVgenome. Fig. 4 illustrates the restriction endonuclease mapsfor the Towne strain of HCMV (kindly provided by R. La-Femina and G. Hayward). The location of the cloned frag-ment has been determined on the basis of these maps. The800-bp gene fragment has been located in fragment A in theEcoRI map, in fragment P in the BamHI map, and in frag-ment C in the Xba I map. The small 1-kb fragment obtainedin the HindIII and BamHI/HindIII digests has been local-ized at -0.507 map unit between fragments H and N in theHindIII maps (Fig. 4).

DISCUSSIONHCMV has been shown to undergo regulated phases of tran-scription and translation giving rise to immediate early, ear-

ly, and late transcripts (36, 37). Although some of the imme-diate early viral proteins may have regulatory functions nec-essary for efficient transcription (38), the late transcriptscomprise largely the structural polypeptides (36). Severalgroups have reported the cloning of restriction fragments of

HCMV in plasmid (39-42) and cosmid vectors (43). This hasprovided a basis for constructing cleavage maps and deter-mining the structural organization of the HCMV genome.Also the transcription pattern of HCMV genome has beeninvestigated at various phases of infection (24, 37).To correlate a defined virion protein with the respective

coding sequence, we have utilized an approach that is based

A1 9 A A

B1 2 3 4

_ -30

23

6.15

1.0

FIG. 3. Hybridization of the cloned 800-bp gene fragment codingfor HCMVgp64 to the restriction fragments of HCMV (Townestrain). HCMV DNA was digested with restriction endonucleasesEcoRI, BamHI, HindIII, and Xba I. (A) The restriction fragmentswere subjected to electrophoresis on 0.7% agarose gels and visual-ized with an ethidium bromide stain. (B) The DNA fragments weretransferred to nitrocellulose by Southern blotting and hybridized to32P-labeled purified 800-bp cloned DNA. The filters were washedsequentially as follows: 0.15 M NaCI/0.015 M sodium citrate/0.1%NaDodSO4, 30 min at room temperature; 15 mM NaCI/1.5 mM sodi-um citrate/0.5% NaDodSO4, 2 hr at 68°C. After washing, the filterswere subjected to autoradiography. Lanes: 1, HCMV-EcoRI digest;2, HCMV-BamHI digest; 3, HCMV-HindIII digest; and 4, HCMV-Xba I digest. Approximate sizes in kb of hybridizing fragments areindicated to the right.

Tyr AriTAC CGG

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CMV Towne

Xbo I

Hindm

BomHZ

EcoRI

M1 Os A L K P B J C R N E TOM2:H G U I

K, 0 M U L I BXJ 0 A H 8NY O? C ZWK2PTRS F V.~~~~~~~~~~~~ . .I I14 ..+..4.-.....--.. .-

Z1S, G ?GY A F ? E R K C BFV P WX?O D MdT JUS2 L B O N

UOl4 F G XdS?NAAT D K OV E W A Y B Cd)PeF I R?0y2 Mc! c J z

-~ L -.:-. S ~

D: MHF:MI

E: KP ()?G:KV

H:ZL E)I:ZN

H:UML:UZ

0 0.1Map Units i

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0I I I I I I I I

FIG. 4. Mapping of the gene fragment coding for HCMVgp64. The restriction endonuclease cleavage maps of HCMV (Towne strain)genome were kindly provided by R. LaFemina and G. Hayward. Based on the results of the Southern blot analysis shown in Fig. 3, the mappositions of the cloned gene fragment have been determined. The map locations are assigned in the A fragment of HCMV-EcoRI, P fragment ofHCMV-BamHI, and C fragment of HCMV-Xba I maps and are shown by bracketed underlines. More precisely, it has been assigned a positionbetween fragments H and N indicated by an arrow in the HCMV-HindIII maps.

on protein sequence information. Synthetic oligonucleotidesmodeled after the amino acid sequences ofHCMVgp64 wereutilized for the probing of a library of HCMV subgenomicDNA. Recent advances in protein purification (19, 44) andmicrosequencing (21) have made this approach quite gener-al, relatively rapid, and applicable to many viral proteins.We have evaluated two oligonucleotide probes 17 bases

and 20 bases long because we were not certain what length(and sequence complexity due to codon degeneracy) wouldbe necessary for optimal screening efficiency. The use ofshorter probes up to 14 bases long is known to give frequentfalse positives, in which the clones have only partial nucleo-tide matches with the probe (45, 46). The use of either twooverlapping probes or a second probe directed against a dif-ferent site in the protein has been suggested to overcome thisproblem (46, 47). Our results have demonstrated that a mix-ture of 16 heptadecanucleotides and a mixture of 32 icosanu-cleotides are similar in terms of giving strong signal-to-noiseratios in both colony screening and plasmid DNA screening.Evidence that the cloned DNA contains sequences coding

for HCMVgp64 was provided by comparing nucleotide se-quences with amino acid sequences of tryptic peptides. Thestrategy that we used to obtain such information is as fol-lows: we determined amino acid compositions of all of thepurified tryptic peptides produced from HCMVgp64. Ofthese peptides, we initially selected T-14 for microsequenc-ing. Amino acid sequence analysis of this peptide gave a sin-gle sequence through the COOH terminus and provided in-formation for the design of synthetic probes. The entire ami-no acid sequence of this peptide was later revealed to havecomplete agreement with the nucleotide sequence of a por-tion of the cloned DNA, thus providing evidence that thecloned DNA coded for HCMVgp64. To further confirm this,we identified another tryptic peptide, T-12, which, on thebasis of amino acid composition, corresponded to a regionlocated 3' to the coding sequences of T-14. When subjectedto microsequence analysis, this peptide also yielded a uniquesequence and showed a perfect match with the amino acidsequence deduced from the nucleotide sequence. These re-sults provide heretofore unreported information on the ami-no acid and gene sequences of HCMVgp64. Identification of

the nucleotide sequences coding for proteins of HCMVshould form the basis for the design of synthetic peptide im-munogens.Very little has been known in terms of precise map posi-

tions of the genes encoding specific HCMV proteins. Stinskiet al. (48) have recently identified the coding region of a 72-kDa immediate early protein by in vitro translation ofmapped mRNA. However, the mapping of the structuralproteins of HCMV has not been reported to date. In herpessimplex virus, the use of intertypic recombinants based ontemperature-sensitive mutants has permitted the physicalmapping of a number of structural polypeptides (49, 50).However, in the case of a slowly replicating virus such asHCMV, such an approach has not been feasible. The methodfor mapping that we have described here utilizes a clonedDNA that contains the nucleotide sequences coding for a de-fined virion polypeptide and thus is a more direct route tothe mapping of specific proteins. Using this approach wehave been able to characterize and physically map the cod-ing sequences of HCMVgp64 in an area of HCMV genomethat has been observed not to be homologous to cellulargenes (unpublished results).We have shown previously that HCMVgp64 contains

-2.34% (wt/wt) galactosamine (19). The exact function ofthis protein and a uniform nomenclature have not been es-tablished. However, it has been suggested that this is a ma-trix protein based upon parallel studies with the Colburnstrain CMV, a simian virus (51, 52). Late in infection thisprotein is overproduced and forms >90% of the protein massof HCMV dense bodies, which are membrane bound, DNA-free, protein aggregates. The antigens conventionally usedfor HCMV serologic testing are rich in HCMVgp64, andantibody to this protein occurs after natural infection (15,18). The role of HCMVgp64 during natural infection is notknown, but it is possible that if overproduction of this mate-rial occurs in vivo, it will have some influence on pathogene-sis of HCMV disease.The cloning of the gene for HCMVgp64 should make it

possible to produce, by chemical synthesis or by expressionin prokaryotic or eukaryotic cells, polypeptides containingthis sequence and variations of it. The expressed proteins

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should provide material that will be useful as a laboratorydiagnostic reagent and may form the basis for the future de-velopment of an HCMV vaccine.

We gratefully acknowledge R. L. LaFemina and G. S. Haywardfor sharing information on the restriction enzymne maps of theHCMV genome and T. Hunkapiller for providing computer pro-grams for DNA sequence analysis. This research was supported byGrant CA 30206 from the National Cancer Institute, Core Grant CA33572 from the National Institutes of Health, and the Harold "Bud"Foley and Charles B. Kaiser, Jr., Research Fund.

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