Structure and complete nucleotide sequence of the Marek's disease herpesvirus gp57-65 gene.

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Vol. 62, No.7 JOURNAL OFVIROLOGY, JUlY1988, p. 2373-2379

0022-538X/88/072373-07$02.00/0

Structure and Complete Nucleotide

Sequence

of the

Marek's

Disease

Herpesvirus gp57-65

Genet

PAUL M. COUSSENSt* AND LELAND F. VELICER

Department ofMicrobiology and Public Health, Michigan State University,EastLansing, Michigan 48824 Received 9 December1987/Accepted 17 March 1988

The gene encoding the precursor polypeptide of the Marek's disease herpesvirus (MDHV) secretory

glycoprotein gp57-65 (formerly identifiedasAantigen) has been sequenced.Previous results had localized the

genetoa4.6-kilobase (kb)segmentof the BamHI Bfragmentintheunique long regionof the MDHVgenome.

S1 nuclease protection experiments were used tomore precisely locate the 5' initiation and approximate 3'

termination points of the approximately 1.8-kb MDHV gp57-65 mRNA within this segment. These results indicated that the entire MDHVgp57-65codingsequenceis contained withina2.35-kb PvuII-EcoRIfragment,

with the direction oftranscription from PvuII to EcoRI (5'to3'). Nucleotidesequenceanalysis of this region revealedasingleopenreading frame of 1,515 base pairs. The MDHV gp57-65 codingsequencehasanoverall

guanosine-plus-cytosine content of 41 %. Translation of the single open reading frame would produce a polypeptideof 505 aminoacids, withacalculated molecularweightof56,805.Theputative gp57-65precursor

polypeptide contains features common tomany glycoproteins. These includea hydrophobic amino-terminal region (aminoacids1to27) thatmayfunctionas asignal peptide and nine potential N-linked glycosylation sites (Asn-X-Ser/Thr). These two features, predicted from nucleotide sequence data, are consistent with the

published data showing that gp57-65 has a signal peptide and N-linked glycosylation (R. J. Isfort, R. A. Stringer, H.-J. Kung, and L. F. Velicer, J. Virol. 57:464-474, 1986). The predictedsequenceindicatesthat overall the polypeptide is relatively hydrophobic, with a possible 18-residue carboxyl-terminal membrane

anchorsequence. Thissequenceappearstobe less prominent than thosecommonly found in integral membrane glycoproteins. The lack of a strong hydrophobic anchor sequence may help to explain the predominantly secretory natureof MDHVgp57-65.

Marek's disease herpesvirus (MDHV) is the causative agentof Marek'sdisease (MD),alymphoproliferative

disor-der of chickens which results in T-cell lymphomas and

peripheralnervedemyelination (11, 14,15). Anantigenically related apathogenic herpesvirus of turkeys (HVT) has been used as a successful live-virus vaccine which prevents the symptoms ofMD (13) but does not prevent MDHV infec-tion. HVT represents the first successful vaccine against a naturallyoccurring tumor in any species. Thus, the MDHV-HVT system provides a unique natural host animal model system for the study of the immunoprevention and the

pathogenesis ofan oncogenic herpesvirus disease.

To more completely understand the nature of HVT-in-duced immunity to MDHV symptoms, we and others have

investigated antigens common toboth viruses (8, 9, 13, 17, 19, 23, 24). At least sixvirallyencodedantigenic proteins are

common between MDHV and HVT (4, 22). One of these proteins, previously designated the A antigen (2, 5, 8, 9), is

a predominantly secreted glycoprotein with an apparent molecularweightof 57,000to65,000 in itsfully glycosylated form (6), hereafter referredto asgpS7-65. Acombination of pulse-chase and cell-free translation studies have shown that

gp57-65 isfirst synthesized as aprecursorpolypeptide with

an apparent molecular weight of 44,000 (pr44). Previous estimates of molecular weightwere based on an ovalbumin marker size of 46,000 daltons and indicated that the size of thegp57-65precursorpolypeptide was 47,000 daltons (6). A

*

Corresponding

author.

t Article no. 12456 of the Michigan Agricultural Experiment Station.

tPresent address:Departmentof AnimalScience,MichiganState University, EastLansing,MI 48824.

later revision of the ovalbumin marker size to 43,000 daltons suggests that the apparent molecular mass of the gp57-65 precursorpolypeptide is, infact, 44,000 daltons.

Aftercleavage ofasmallsignalpeptide, the polypeptideis

glycosylated and secreted from the cell in a precisely pro-grammedmanner(6). Althoughgp57-65 isoneof theprimary proteins recognized by sera from MDHV- or HVT-infected chickens,itspreciserole,ifany,intheimmunopreventionor

pathogenesis ofMD is unclear. It has been postulated that the extensively secreted gp57-65 may play an

immunoeva-siverole in the pathogenesisof MD(6), and it isacandidate for causing the early-stage immunosuppression that occurs after MDHVinfection(5).

Recently, the gene encoding the precursorpolypeptideof MDHVgp57-65 waslocalized to a4.6-kilobase(kb) PvuII-SmaI segment of the BamHI Bfragment(5), with reference

to the restriction map of the MDHV genome generated by Fukuchietal. (1). In the present report, we describea more

precise localization of the MDHV gp57-65 gene, presentits

complete nucleotide sequence, and analyze its predicted precursorpolypeptide, especiallyin relation to known prop-erties of the antigen.

MATERIALS ANDMETHODS

Cell cultures and viruses. The preparation, propagation, and infection of small-scale duck embryo fibroblast (DEF) cell cultures with MDHV were performed as described

previouslybyGlaubigeretal. (2) andmorerecently by Isfort

etal.(5). MDHV strain GAwasusedatpassage level 6after

itwasisolated from feathertipsobtainedfrom infected birds (21).

Restriction enzymes, generation of M13 subclones, and nucleotide sequencing. The MDHV (strain GA) BamHI

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2374 COUSSENS AND VELICER

brary was a gift from M. Nonoyama, Showa University Research Institute (1). The MDHV DNA in each clone was propagated in Escherichia coli DH-5 and was extracted by standard methods (10). All restriction and modifying en-zymes werepurchased fromeither Bethesda Research Lab-oratories, Inc. (Gaithersburg, Md.) or Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and used according to the instructions of the manufacturer. In preparation for nucleo-tide sequencing, the 2.35-kilobase-pair (kbp) PvuII-EcoRI segment of the BamHI B fragment was subcloned into plasmid pUC19 cleaved with SmaI and EcoRI to generate plasmid p19MDA2.35 (ATCC 40312). This cloning

proce-dure destroyed the PvuII site of the MDHV fragment but placed a convenient BamHI cleavage site from the multiple

cloningregion of pUC19 4 nucleotides 5' of theSmaI-PvuII

junction. Subclones of MDHV DNA were prepared in

M13mpl8-M13mp19 vectors by standard techniques (10).

Single-strand recombinant M13 DNA templates were

pre-pared by polyethylene glycol precipitation of virus from culture supernatants andextraction with phenol and

phenol-chloroform(1:1), followed byethanol precipitationof single-stranded viral DNA. Templates were sequenced by the

dideoxy chain-termination method (20), using the Klenow fragment of DNA polymeraseI,[a-32P]dATP,and an appro-priate ratio of dideoxynucleotides to deoxynucleotides.

Products of sequencing reactions were analyzed on 8% polyacrylamide gels containing8 M urea.

IsolationofcellularRNA.TotalcellularRNAwasisolated

from normal and MDHV-infected DEF cells essentially as

described previously (10). Briefly, cells were lysed in 4 M

guanidinium isothiocyanate. DNA wassheared by repeated

passageofthe celllysates through an18-gauge syringe. The final preparation was loaded on a cushion of5.7 M CsCl in 0.1 M EDTA. Centrifugation was carried out at a force of

174,000 x g for 16 h. RNA pellets were solubilized in a

solutioncontaining100 mMTrishydrochloride(pH 8.0) and 10 mM EDTA and extracted twice with phenol-chloroform

(1:1) and once with chloroform. After precipitation with

ethanol, RNAwassolubilized inwater at aconcentration of 5 Lg/,uland storedat -70°C untiluse.

Si

nucleaseprotection studies.Nucleaseprotection studies

were performed essentially as described elsewhere (10),

usingtheprobes described in thetext. Hybridizations were

routinelyperformedat50°Casrecommended forDNA, with

an averageG+C content ofapproximately 46% (10).

a P/SI

B} 1K

I

K BI

Kb 00 1.0

S\N

6

sv 20

1

2.0 E

I I I

ru

b 2 3 4 5

234

6 7 8 Size(bp)

- 1,353

* - 622

-527 - ~-404 - 309

.242 -238 -217

-201

-

180

FIG. 1. Finestructuremapping oftheMDHVgp57-65gene. The orientation andlimitsoftheMDHVgp57-65gene weredetermined by

Si

nuclease protection experiments, using probes I and 11 (a). Probestodeterminetheorientation and5'limitswere5'endlabeled with [-y-32P]ATP, using T4 polynucleotide kinase. After denatur-ation, hybridization to RNA, and S1 digestion, protected species wereanalyzedon5% denaturingpolyacrylamidegels(b). Lanes:1, Probe 11withoutRNAand S1; 2, probeI withoutRNAand

Si;

3, probe II plus MDHV-infectedDEFRNAandS1 nuclease; 4,probe 11 plus uninfected DEF RNA and Si nuclease; 5, probe I plus MDHV-infected DEF RNA and

Si

nuclease; 6, probe I plus uninfectedDEF RNA and

Si

nuclease;7and8;sameaslanes 5and 6,respectively,except that thehybridizationtemperaturewas45°C instead of50°C.Todeterminetheapproximate3'limitofthe MDHV

gp57-65 gene (panel a, dashed arrow), probe I1 was labeled with

[(a-32P]dGTP,

usingtheKlenowfragment ofE. coliDNA polymer-ase 1. Theapproximate5' initiation and 3' termination sites ofthe MDHVgp57-65mRNAareindicatedinpanelaby solid and dashed arrows, respectively. Restriction enzymes used in subcloningand thegeneration of probeswereBamHl(BI),Bglll(BII), KpnI (K),

Sspl (S), NcoI (N), EcoRV (V), EcoRI (E), PvuII (P), andSmaI

(SI).

RESULTS

Precise mapping of the MDHV gp57-65 gene. Previous

results hadlocalized the MDHV gp57-65 (previous nomen-clature, MDHV A antigen) gene to a 4.6-kb PvuII-SmaI segment of the BamHI B fragment but had not precisely mapped the gene or determined its orientation 5'->3' (5). Beforenucleotidesequenceanalysis,weattemptedtodefine the5' and3' ends of thegene.Resultsfrom SP6

polymerase-and T7 polymerase-generated single-stranded RNA probes

had indicated that the direction oftranscription was 5'->3' PvuII-+EcoRI (P. M. Coussens and L. F. Velicer,

unpub-lished results). To confirm this result and to locate the

approximate RNAstart site, 5'-end-labeled probes I and II

(Fig. la)wereprepared.Afterdenaturation, hybridizationto MDHV-infected cell RNAoruninfected DEFRNA,and S1

nuclease digestion as described in Materials and Methods,

the protected fragments were sized on a 5% denaturing polyacrylamide gel and detectedbyautoradiography. Probe Iextended from theBamHI sitetotheBglIIsite(Fig. la)and

migratedwithanapparentsize ofapproximately650bp

(Fig.

lb, lane 2). Probe II extended from the BglII site to the

EcoRI site and was

approximately

1,400 bp long

(Fig. lb,

lane 1). After denaturation and

hybridization

to MDHV RNA, two segmentsofprobe

I,

a major

species

of approx-imately 486 nucleotides and a minor species of

approxi-mately 364 nucleotides, were protected from S1 nuclease

digestion (Fig.

lb,

lane 5). As expected, uninfected DEF RNA was unable to protect probe I from S1 nuclease

digestion (Fig. lb, lane 6). Identical results were obtained

with probe I when the hybridization temperature was

re-duced to 45°C (Fig.

lb,

lanes 7 and 8). These results suggested that two possible transcriptional initiation sites existfor the MDHV

gp57-65

gene andconfirmedthe5'->3'

orientation

predicted

by the SP6 polymerase- and T7

poly-merase-generated RNA probes. In addition, these results

indicated that the 5' end of the MDHV

gp57-65

mRNA

mappedatmost486nucleotides5' of the

BglII

site. ProbeII was completely digestedby

Si

nuclease in the presence of

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STRUCTURE AND SEQUENCE OF THE MDHV gp57-65 GENE

(Kbp)0

B T

I I

0.5

T T g T

I aI

1.0 T

1.5 20

TN T

0 .- it

2.5

TR E

.1 I

-4 __

4t-c .

*e

4~ ~ _

FIG. 2. Strategy of andclones prepared for nucleotide sequence analysis of the 2.35-kbPvuII-EcoRI fragment of the MDHV genome containing all of the MDHV gp57-65 coding region, determined by S1 nuclease digestionasdescribed in thelegendtoFig. 1.All cloneswere prepared in eitherM13mpl8orM13mpi9asappropriate. Arrowsindicate thedirection andextentofDNAsequenceanalysison aparticular fragment.Restrictionenzymesites shownareBamHI(B),BgIII(Bg),NcoI (N), TaqI(T), EcoRV (R), and EcoRI (E).

MDHV-infected cellRNA oruninfectedDEF RNA(Fig.lb, lanes 3 and 4, respectively), asexpected from our previous

results and theprotection of probe I in thepresent study. TheMDHVgp57-65 mRNA has been shown by Northern

(RNA) blot analysis to be approximately 1.8 kb long (5; P. M. Coussens and L. F. Velicer, unpublished results). Theseresultscombinedwith the 5'Simapping data strongly

suggested that the MDHV gp57-65 3' terminus was within

the 2.35-kb PvuII-EcoRI segment. However, the results of

Isfort et al. (5) indicated that the adjacent 2.4-kb

EcoRI-SmaI fragment could hybrid select gp57-65 mRNA and

hybridizeto asmallportion of gp57-65mRNAon Northern

blots (5).To morepreciselydefine the 3'limit ofthe MDHV

gp57-65 mRNA,Si nuclease protection studies using

3'-end-labeled probes I and II (Fig. la) were performed. As

ex-pected, probeI, whichwaslabeledonthecoding strandfor

3' endmapping, was notprotected from Si nuclease diges-tion by hybridization with MDHV-infected cell RNA (data notshown). However,fragments of probeII wereprotected (data not shown), which suggests that the 3' end of the

gp57-65 coding region was contained entirely within the

2.35-kb PvuII-EcoRI fragment. These results (Fig. la, dashed arrow) were in agreement with the interpretation obtained bycombining the 5'

Si

mapping data (Fig. 1)and datafrom Northern blots probed withsubfragments ofthis

region (Coussensand Velicer, unpublishedresults). Nucleotide sequence of the MDHV gp57-65 gene. The results obtained by

Si

nuclease protection analysis had

localizedthe MDHVgp57-65gene within the 2.35-kb PvuII-EcoRIsegment oftheBamHIB fragment. As apreliminary

step to nucleotide sequence analysis, an ordered set of

M13mpl9subclones for thisregionwasprepared by cloning

TaqI

digestion products intothe AccIsite ofM13mpl9. The

preparation ofadetailed TaqI restrictionmapofthegpS7-65 coding regionalso allowedrapidcomparison ofthegpS7-65

genesisolated from variousMDHVandHVTisolates(P. M.

Coussens, R. A. Stringer, and L. F. Velicer, unpublished

results). Severalclones representing each of the TaqI frag-ments wereisolated. Nucleotide sequence analysisof these

clones allowedrapid bidirectional sequencing ofmostof the

2.35-kb PvuII-EcoRI segment (Fig. 2). Junctions were

se-quencedfromadditional clones generatedbyeitherBAL31

digestion from specific sites or directed cloning in M13 vectors with the restrictionsites indicated in Fig. 2. Terminal sequences were determined by cloning the entire 2.35-kb

fragment into both M13mpl8 and M13mpl9. Since this

represented the first MDHV gene to be sequenced in its

entiretyand it would be used as a standard forcomparison

with the

gpS7-65

genesofother MDHV strains and serotypes

(including

HVT), many regions were sequenced several times in bothdirections (Fig. 2).

The complete nucleotide sequence ofthe region

contain-ing the putative gpS7-65 coding region is shown in Fig. 3. There is asingle large openreading frame within this region extending from the ATG codon beginning307 bp 3' ofthe

BamHI site (nucleotide 1 in Fig. 3) ofp19MDA2.35 to the

termination codon TAG, startingatnucleotide1516ofFig.3.

Translationofthis 1,515-bpopenreading frame (Fig. 3,line

2) would produce a polypeptide of 505 amino acids, with a

calculated molecularweight of 56,805. There arefour poten-tial TATA sequencesinthe307bpimmediately upstreamof

the MDHVgpS7-65 initiation ATG. These occur at

positions

-49 to -52, -190 to -193, -239 to -242, and -276 to -279. There are threesequences which may act aspotential CAT-box regions, ranging from nucleotides -94 to -100, -161 to -172, and -207 to -219. The sequence AATAAA

from -157 to -163 represents a possible polyadenylation signal fora gene 5' to the MDHV gpS7-65gene. The same AATAAA sequence occurs onceafter thegpS7-65 termina-tioncodonbetweennucleotides1655 and 1661 (not shown in

Fig. 3) and is the probableMDHV

gpS7-65

poly(A) addition signal. Consistent with buoyant density measurements of

total MDHV DNA (7), the overall guanosine plus cytosine (G+C) contentofthe

gpS7-65

coding regionis 41.6%.

The predicted amino acid sequence of the precursor

MDHV

gpS7-65

polypeptide, translated from the DNA se-quence,exhibits featurescommon toother

glycoproteins.

A sequence of 19 hydrophobic amino acids (Fig. 3) in the amino-terminal region may function as part ofthe

gpS7-65

signal peptide (5). This region ends in a sequence that appears tofollow the rulesof Pearlman and Halvorson (16)

forpredicting signal peptide cleavage sites. Inthis case, the

peptide cleavage site would be the alanine (amino acid 28) whosecodon begins at nucleotide 82 (Fig. 3). There are also

ninepotentialN-linkedglycosylationsites(Asn-X-Ser/Thr),

which areunderlinedin Fig. 3.

Analysis ofthehydrophilicity ofthepredicted amino acid

sequence (Fig. 4) indicates that, overall, the protein is

relatively hydrophobic and contains an 18-residue (amino acids 461 to 479) hydrophobic sequence in the carboxyl terminus.Despite thepredominantly secretory natureofthe

gpS7-65

polypeptide, thisregion orothers of similar hydro-phobicity could function as possible membrane anchor se-quences similar to those found in glycoproteins which are known to interact with the plasma membrane. The

hydro-phobic sequence representing the putative

gpS7-65

signal peptide, residues 1 to 27, is clearly visible in this analysis (Fig.4). Consistentwith the hydrophilicity plot (Fig.4), the codon usage table(Table1) shows that thepredicted

gpS7-65

aminoacid sequencecontains 55.4%nonpolaramino

acids,

including tryptophan (8.6%) and isoleucine

(7.2%)

as the most abundant amino acids in the sequence.

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-300 -290 -260 -270 -260 -250

* * * * * *

OCA TCC CCC TCC CTA TCT ACT CAAATC TAT ACC TCG CAT TTA TCA TAA CTA CTT TAC TTO

-240 -230 -220 -210 -200 -190

* * * * * *

Tm CTA TAT TCA CTA CCC CTA TCT TOA CCAMT COTTCT TCA CAT CTS CCC CAT ATA COT

-180 -170 -160 -150 .140 -130

* * * * * *

ATT OAT COT TCT TTC CAA COO COA ASAMA CTI TCA TAC ATA CTAMCOATCTOA OTT

-120 .110 -100 .90 -80 -70

* * * * * *

TIT TAT GAG COT TCA MA CAA AGO TAC CAT COO TTT AM ACT MC 170 CAT ATC CTA ATC

-60 -50 -40 -30 -20 -10

* * * * * *

CAC MA MT CAT TTT ATA CTCAT CCC CM GA A CCAMC OTA ACC CTC TAC ATA TCT

1 10 20 30 40 S0

* * * ,

-*---4---*-____-TCC CTCATO CTC AGO CCOC COT OT TA CCA OCT TIC0OC TOG ACT COA CTC TTT Tm TTC Not Lou Thr fto Arg Va1 Lou ArgAl Lu OlyTrp Thr Cly Lou Ph. Ph. Lou

60 70 t0 90 100 110

~~- ---* * * *

CTT TTA TCT CCC ACMC OTC CT OGA OCC ACC CTTACC OG CAT CTC CAA CAC CCC ATT Lou Lou Sor tro Ser As Va1 tLu Oly Ala Sr Lou Ser

Arg

Asp Lou Clu His Pro Ile

120 130 140 1S0 160 170

* * * * * *

CTA TCC TIT CAT CCA TCC MC ATT TC ATT MCC¢C OCC CCT TIAACT GAG CO CCT CAT Lou S.r Ph. Asp Pro Ser AwnlIe Sewr Ile Asa ly Ala Pro Lou Thr GluVa1 Pro His

1S0 190 200 210 220 230

* * * * * *

CCA CCT TCC AC CM ACT OTC TCA ACA MTTCO CMA AGTACC MT CM CATACC ATA ACA Ala fto SrThr Clu SerVa1 Ser Thr Asa Ser Clu Ser Thr As. Clu His Thr IlI Thr

240 250 260 270 280 290

* * * * * *

CM ACO ACO 0C0 MC MC OCA TAC ATC CAC MC MTCC TCT ACO CAC MC CM MT CCC Clu Thr Thr Oly Lys Asn Ala Tyr Ile His s Asa Al Ser Thr Asp Lys Gln As. Ala

300 310 320 330 340 350

* * * * * *

MC OAC ACT CAT MAA ACO CCC MT ATA CTC TOC CAT ACO GM CM G01 117 01 TTC CTT AsnAsp Thr His Lys Thr Pro Asn Ile Lou Cys Asp Thr Clu Clu Val Ph. V-1 Ph. Lou

360 370 380 390 400 410

* * * * * *

MC GM ACG GGA mA TTT GTT TOT ACT CTC MA C01 OAC CCC CCC TCCOAT ACT CM TOG AsnGlu Thr Gly Ar; Ph. Va1 Cys Thr Lou Lys Va1 Asp Pro Pro Ser Asp Ser CluTrp

420 430 440 450 460 470

* * * * * *

TCC MC TTT GTT CTA GAT CTG ATC Tm MC CCA ATT GM TAC CAC CCC MC CM MC MT Ser Asn Phe Va1 LouAsp Lou Ile Ph. Awn Pro Ile luTyr His Ala Awn Glu Lys Asn

480 490 500 510 520 530

* * * * * *

T0 GCM GCG GCG CGT ATC GCT GGT CTC TAT GGA GTC CCC OCA TCA CAC TAT GCA TAC CCA Va1 Glu Ala AlaAr; Ile Ala Gly Lou Tyr GlyV.1 Pro Gly Ser Asp TyrAla lyr Pro

540 550 560 570 580 590

* * * * * *

COT CM TCT CM TTA ATT TCT TCC ATT CGA CGA OAT CCC CAC C ACA TT1 TOGACC AMC

Arg Cln S-r Clu Lou Ile Ser S.r IleArg Arg Asp Pro Gln Gly Thr Ph. Trp Thr Ser

600 610 620 630 640 650

* * * * * *

CCA TCA CCT CAT GGAMC AMG TAC TTC ATA TOG ATAAMC MA AGA ACC MT ACO ATO0CC

fto S.r Pro His Gly Awn Lys Tyr Ph. Ile Trp Ile Asn Lys Thr ThrAwn Ther Ht Gly

FIG. 3. DNA sequenceof theMDHVgp57-65gene. The sequenceis numbered with+1asthefirstbaseoftheinitiationATG codon of the 505-amino-acid open reading frame. The predicted aminoacid sequence of thegp57-65 precursor polypeptide is written below the nucleotide sequence,arrangedascodonsin segmentsofthreenucleotides. Thehydrophobicamino-terminalsequence that mayfunctionas

a signal peptide is highlighted by adashed line above the sequence (nucleotides +25 to +81). Potential sites ofN-linked

glycosylation

(Asn-X-Ser/Thr)areunderlined.

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660 670 680 690 700 710

* * * * * *

GTG GAA ATT AGAMT OTA GAT TAT GCT GAT MT OGC TAC ATG CM GTC ATT ATG CCT GAC Val Glu Ile Arg AsnVal Asp Tyr Ala Asp Asn Gly TyrHot Gln Val Ile Hot Arg Asp

720 730 740 750 760 770

* * * * * *

CAT TTT MT COG CCT TTA ATAOAT AA CATATE TAC ATACOT OTO TOT CM CGA CCTOCA His Ph. AsnArg Pro Lou Ile Asp Lys His Ile Tyr Il Arg Val Cys Gln Arg ProAla

780 790 800 610 820 830

* * * * * *

TCA GTG GAT GTA CTO GCCCCT CCA GTC CTC AGCGGA GM MT TAC MO GCA TCT TOT ATC Sor Vol Asp Val Lou Ala Pro Pro Val Lou Sor GlyGluAsn Tyr Lys Ala Ser Cys Ile

840 850 860 870 880 890

* * * * * *

GTT AGA CAC TEE TAT CCC CCT GGA TCT OTC TAT OTA TCT TGG AGA CAG MT GGA MC ATT Vol Arg His Ph. Tyr Pro Pro Gly Ser Vol Tyr Vol Ser Trp ArgGln Asn Gly Asn Ile

900 910 920 930 940 950

* * * * * *

GCA ACT CCT COG AMA AT COC OAT GGA ACT TET TGG TOG TTC GM TCT GGT AGA GGA GCT Ala Thr Pro Arg Lys Asp Arg Asp Gly Ser Ph. Trp Trp Ph. Glu Ser Gly Mrg Gly Ala

960 970 980 990 1000 1010

* * * * * *

ACG TTG GTT TCT ACA ATA ACA TTG GGA MT TCA GGA ATT OAT TTC CCC CCC MAATA TCT

Thr LouVal Ser Thr Ile Thr Lou GlyAsn Ser Gly Ile Asp Phe Pro Pro Lys Ile Ser

1020 1030 1040 1050 1060 1070

* * * * * *

TGT CTG GTT CCC TGG MG CAG GOT OAT ATO ATC AGC ACO ACO MT GCC ACA GCT ATC CCG Cys Lou Val Ala Trp Lys Gln Gly Asp Het Ile Ser Thr Thr JAs Ala Thr Ala Ile Pro

1080 1090 1100 1110 1120 1130

* * * * * *

ACG OTA TAT CAT CAT CCC CGT TTA TCC CTG GCT TTT MA GAT GGG TAT GCA ATA TGT ACT Thr Vol Tyr His His Pro Arg Lou Ser LouAla Ph. Lys Asp Gly Tyr Ala Ile Cys Thr

1140 1150 1160 1170 1180 1190

* * * * * *

ATA GM TOT GTC CCC TCT GAG ATT ACT OTA CGG TGG TTA GTA CAT GAT GM GCG CAG CCT Ile GluCys Vol Pro S-r Glu Ile Thr Val Arg Trp LouVal His Asp Glu Ala Gln Pro

1200 1210 1220 1230 1240 1250

* * * * * *

MC ACA ACT TAT MT ACT GTO GTT ACA GOT CTC TGC CGG ACC ATC GAT CGC CAT AGA MT Asn Thr Thr Tyr Asn Thr Vl Vol Thr Gly Lou Cys Arg Thr Ile Asp Arg His Arg Asn

1260 1270 1280 1290 1300 1310

* * * * * *

CTC CTC AOCCGC ATT CCA OTA TOG OAC MT TOG ACG AA ACA AMTAT ACO TGC AOA CTC Lou Lou Ser Arg Ile Pro Val Trp Asp An Trp Thr Lys Thr Lys Tyr Thr Cys Arg Lou

1320 1330 1340 1350 1360 1370

* * * * * *

ATA 0GC TAC CCC TTC GAT GM GAT MA TET CM OAT TCG GM TAT TAC OAT GCA ACT CCA Il Oly Tyr Pro Ph. Asp Glu Asp Lys Ph. Gln Asp Ser Glu Tyr Tyr Asp Ala Thr Pro

1380 1390 1400 1410 1420 1430

* * * * * *

TCT GCA AOA GGA ACA CCC ATO GTT ATT ACG GTT MC 0C0 ACT TEE 0G0 ATT GGC TGT MT Ser Ala Arg Oly Thr Pro Met Val Il. Thr Val Asn GI Ph. Gly Ile Gly Cys Asn

1440 1450 1460 1470 1480 1490

* * * * * *

TET AGO GTA GOG GAT MT CAT GAC TGC CCT ATO TmT ATA CAC TCC ACA COA AM ATA TTC Ph Arg Vol Oly Asp Asn His Asp Cys Pro Hot Ph. Ie His Sor Thr Arg Lys Ile Ph.

1500 1510 1520 1530 1540 1550

* * * * * *

OAT TAT MT CTC ATT GTTATO TAG TTG TGA TmT ATT MA CAT ATT TET TAT MC TCT ACT Asp Tyr Asn Lou Ile Vl Hot

---1560 1570 1580 1590 1600 1610

* * * * * *

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ATT CTC CGA GTE ACT TAT ATA TET ATE TOT CAG ACA ATA ATC CM TAG TGG AA MTCOT FIG. 3-Continued.

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2378 COUSSENS AND VELICER

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I 100.8 200.8 300.4 400.2 500

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SEQUENCE POSITION

FIG. 4. Hydrophilicity plot of the predicted MDHV gp57-65 aminoacid sequence.The plotis basedonthealgorithmofHopp andWoods(3)forpredictingpossibleantigenic determinantsites.

DISCUSSION

The MDHV gp57-65genehas beenlocalized, molecularly

cloned, and sequenced. The results presented in this report show that the gp57-65geneis located nearthecenterof the BamHI B fragment and is contained entirely within the 2.35-kb PvuII-EcoRI fragment subcloned into plasmid pUC19togenerateplasmidpl9MDA2.35. Molecular hybrid-izations and nucleotide sequence analyses have indicated

that the analogous gene from the immunologically related vaccinevirus,

HIVT,

islocated inanidentical position in the HVTgenome(Coussens and Velicer, unpublished results).

Nucleotidesequenceanalysisof the cloned 2.35-kb

PvuII-EcoRI segment of the BamHI B fragment revealed a long

openreading frame of 1,515 nucleotides which, when trans-lated, encodes a polypeptide of 505 amino acids, with a calculatedmolecular weight of 56,805. The predictedMDHV

gp57-65 polypeptide contains features common to other

known glycoproteins and consistent with previously re-ported properties of the protein (6). One such property is a

hydrophobic amino-terminal region (amino acids1to27)that endsinasequenceconsistentwith the rules of Pearlman and

Halvorson for predicting glycoprotein signal peptidase cleavage sites (16). The existence of a signal peptidase cleavage site in the gp57-65 precursorwas expected, since

incubation of the gp57-65 precursor synthesized in cell-free translation reactions with dog pancreatic microsomes re-sulted incleavage of the precursor(6). The precise site of

signal peptidase cleavage is being investigated by N-terminal

sequence analysis of the mature gp57-65 antigen isolated from MDHV-infected cells. The existence of potential sites for N-linked glycosylation (Asn-X-Ser/Thr) was also

ex-pected, since tunicamycin,aninhibitor of N-linked

glycosyl-ation, completely inhibits glycosylation of this antigen (6). Thepredicted polypeptide is relativelyhydrophobic, with

a possible carboxyl-terminal membrane anchor sequence

(amino acids 461to479).Additional hydrophobic amino acid stretches occurat positions throughout the predicted poly-peptide sequence (Fig. 4). Although the predominantly secretory nature of MDHV gp57-65 suggests that these

sequencesareinsufficienttoanchor theprotein in theplasma membrane, recent evidence (6; unpublished observations) indicates that a small amount of mature gp57-65 can be

detected in the plasma membranes of infected cells. At the present time, however, it is not clear whether this small amountofgp57-65 isactually anchored in the plasma mem-braneorheld by other interactions.

The predicted molecular weight of the gp57-65 precursor

polypeptide (56,805) is considerably

larger

than theapparent

molcularweight of 44,000 currently estimated from previous sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of immune precipitation products from cell-free translation reactions or pulse-chase studies (6). With this

method, severedistortion ofprotein migrationinthisregion ofthegelmayresultfromthelargeamountsof immunoglob-ulin G heavy chains (Mr -50,000) present in the immune precipitation reactions. Experiments designed to measure

the apparent molecular weight of the gp57-65 precursor

polypeptide by immunoblotting sodium dodecyl

sulfate-polyacrylamide gelsoftunicamycin-treatedMDHV-infected

DEFculturemediumareinprogressand willprovideasize estimate withoutinterferencefromimmunoglobulin G.

TABLE i. Codon usage in thegp57-65 amino acid sequence

CodonAmino Fre- % Codon Amino Fre- Cd Amino Fre- Codon Amino

Fre-acid

quenccy

acid quencyoon acid quency acid quency

TTT F 16 3.2 TCT S 12 2.4 TAT Y 11 2.2 TGT C 7 1.4

TTC F 6 1,2 TCC S 6 1.2 TAC Y 9 1.8 TGC C 4 0.8

TTA L 7 1.4 TCA S 6 1.2 TAA 0 TGA 0

TTG L 4 0.8 TCG S 4 0.8 TAG 1 TGG W 11 2.2

CTT L 3 0.6 CCT P 11 2.2 CAT H 11 2.2 CGT R 6 1.2

CTC L 12 2.4 CCC P 13 2.6 CAC H 5 1.0 CGC R 3 0.6

CTA L 3 0.6 CCA P 7 1.4 CAA Q 5 1.0 CGA R 5 1.0

CTG L 4 0.8 CCG P 3 0.6 CAG Q 4 0.8 CGG R 5 1.0

ATT I 16 3.2 ACT T 10 2.0 AAT N 20 4.0 AGT S 5 1.0

ATC I 7 1.4 ACC T 4 0.8 AAC N 15 3.0 AGC S 7 1.4

ATA I 13 2.6 ACA T 13 2.6 AAA K 11 2.2 AGA R 8 1.6

ATG M 8 1.6 ACG T 16 3.2 AAG K 6 1.2 AGG R 1 0.2

GTT V 11 2.2 GCT A 6 1.2 GAT D 23 4.6 GGT G 4 0.8

GTC V 7 1.4 GCC A 5 1.0 GAC D 7 1.4 GGC G 8 1.6

GTA V 9 1.8 GCA A 9 1.8 GAA E 20 4.0 GGA G 14 2.8

GTG V 7 1.4 GCG A 6 1.2 GAG E 2 0.4 GGG G 4 0.8

J.VIROL.

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STRUCTURE AND SEQUENCE OF THE MDHV gp57-65 GENE

The total G + C content of the gp57-65 coding region is 41%, which is relatively low compared with that of most

alphaherpesviruses, such asherpes simplex virus type1(18), but consistent with that of many othergammaherpesviruses,

such as herpesvirus saimiri (12). Other genes recently iden-tified in the MDHV genome show a slightly higher overall G+C content than the gp57-65 sequence presented in this report (I. Sithole, P. M. Coussens, and L. F. Velicer, unpublished results).

The data presented here represent the first nucleotide sequence of a known coding region within the MDHV genome. Current studies in our laboratory have indicated that while the overall nucleotide sequence of the HVT gp57-65 gene is 80 to 85%homologous tothatof the MDHV gp57-65 gene, most differences occur in the third base position of gp57-65 codons and are thustranslationallysilent (Coussens and Velicer, unpublished data). In addition to

determining the exact relationship between MDHV and HVT, nucleotide sequence analysis ofa growing body of isolated MDHV genes will allow us to examine the regula-torysequences involved in control of MDHV gene transcrip-tion. Itwillbe of particular interesttodeterminetherole of

suchsequences, ifany, intheloss of gp57-65 gene

expres-sion at the transcriptional level after continued passage in cellculture.Experimentstoprecisely locatethe mRNA start

site and to identify the promoter sequences ofthe gp57-65 gene are in progress. Thus, the molecule nowidentified as gp57-65 (formerly called A antigen) continues to be of interest and importance in this natural host animal model system. Furtheranalysis of gp57-65andthe geneencodingit will helpdetermine its precise role in theimmunobiologyand

pathogenesis of MDHV.

ACKNOWLEDGMENTS

This study was supported by grant 85-CRCR-1-1879 to L. F. Velicer under the Competitive Research Grants Program adminis-teredby the U.S. Department of Agriculture and byagrantfrom the Michigan State University Biotechnology Research Center to L. F. Velicer.

We thank Ruth A. Stringer for excellent technical assistance, Peter Brunovskis for help in mapping the TaqI fragments of the gp57-65 gene, Larry Snyder andCheng Kao for helpful technical advice on DNAsequencing, and IdahSithole for careful review of themanuscript and many helpful suggestions.

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