Antigenic and protein sequence homology between VP13/14, a herpes simplex virus type 1 tegument protein, and gp10, a glycoprotein of equine herpesvirus 1 and 4.

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Vol. 65, No.5 JOURNAL OF VIROLOGY,May 1991, p.2320-2326

0022-538X/91/052320-07$02.00/0

Antigenic and Protein Sequence Homology between VP13/14,

a

Herpes Simplex Virus Type

1

Tegument Protein, and

gplO, a

Glycoprotein of Equine Herpesvirus 1 and 4

GARY R. WHITTAKER,' MARCELLO P. RIGGIO,2IAN W.

HALLIBURTON,l

RICHARD A. KILLINGTON,1 GEORGE P. ALLEN,3 ANDDAVID M.

MEREDITH'*

Department of Microbiology, University of Leeds, Leeds LS2

9JT,l

andDepartment of Veterinary Pathology, University of Glasgow, GlasgowG61 IQH,2 United Kingdom, andDepartment of Veterinary Science,

University of Kentucky, Lexington, Kentucky405463 Received 8 November 1990/Accepted 30 January 1991

Monospecificpolyclonal antisera raised againstVP13/14, amajortegumentproteinofherpes simplexvirus type 1 cross-reacted with structural equine herpesvirus 1 and 4 proteins of

M,

120,000 and 123,000, respectively; these proteins areidentical in molecular weighttothecorresponding glycoprotein 10 (gplO) of

each virus. Using a combination of immune precipitation and Western immunoblotting techniques, we

confirmed that anti-VPl3/14 and a monoclonal antibody to gplO reacted with the same protein. Sequence

analysis ofaAgtll insert ofequine herpesvirus 1gplOidentifiedanopenreadingframe inequine herpesvirus 4 with which itshowed stronghomology; this openreadingframe also shared homology with geneUL47 of

herpes simplex virus type 1 and gene 11 of varicella-zoster virus. This showed that, in addition to immunological cross-reactivity, VP13/14and gplO have protein sequence homology; it also allowed identifi-cation ofVP13/14as thegene productof UL47.

The tegument region of the herpes simplex virus (HSV)

particlewas originally definedas anelectron-opaque,

amor-phous region located between the virus envelope and capsid (27); the proteins composing this region were defined by a subtractive method as those not releasedby nonionic deter-gent and not present in the virus capsid. The tegument

contains fivemajor proteins, VP1/2,VP13, VP14,VP16, and

VP22, each of which constitutes -5% of the mass of the virus particle (17, 27).

VP13 and VP14 have been considered to be related throughsomeform ofposttranslational modification (17) and arethoughttobe theproduct ofasinglegene(21a). Thetwo

polypeptides exhibit polymorphismin molecularweight

be-tween HSV type 1 (HSV-1) strains; variation in apparent molecularweight of one polypeptide ismimicked by varia-tion in the molecularweight oftheother (23). All isolatesof

HSV-2 appear to contain a single invariant polypeptide

designated VP14 (5). VP13 and VP14 of HSV-1 have been

shown to be phosphorylated within the virion; the gene

coding for these polypeptides has been mapped to aregion

between mappositions 0.66 and 0.76 on the HSV-1 genome (18).

The studies reported here were carried out as part ofa series of experiments attempting to characterize the tegu-ment proteins of HSV-1, to identify the genes coding for them, and to identify homologous proteins in other herpes-viruses.

MATERIALSANDMETHODS

Cell culture, virus strains, and virus purification. HSV-1 strain SC16 and HSV-2 strain 3345 were cultivated in

*Correspondingauthor.

BHK-21 cells grown in the autoclavable form of Eagle

medium(Flow Laboratories) containing 10%calfserumand 10% tryptose phosphate broth. Equine herpesvirus 1 (EHV-1) strain Ab-1 and EHV-4strain MD were cultivated in either RK13 cells (EHV-1) or NBL-6 cells (EHV-4) as describedpreviously(22). HSV-1,EHV-1, and EHV-4were

purifiedfrom extracellular virusbystandardtechniques (22),

and HSV-2 was purified from cytoplasmic extracts of cells (27).

SDS-PAGEand Westernimmunoblotting.Sodiumdodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and

Western blotting were performed essentially as described

previously (22) with gels containing 9 or 12% acrylamide

cross-linked with N,N'-diallyltartardiamide. Western blots were developed with Vectastain ABC kits and peroxidase-linked antibody (Vector Labs), and color was developed

with

chloronaphthol-H202.

Immuneprecipitation. For immuneprecipitationof

glyco-protein 10 (gplO), RK cells were infected with EHV-1 at 1

PFU/cell and labeled at 6 h postinfection with 10 ,uCi of

[35S]methionine

(Amersham) per ml in medium containing

20% of the normal levels ofmethionine. Cells were har-vestedat24 hpostinfection,solubilized inRIPAbuffer(0.1%

sodiumdodecyl sulfate, 0.5% Nonidet P-40, 0.1MNaCl,50 mMTris-HCl [pH 7.4]), andcentrifugedat100,000 x gfor1 h; the supernatant was removed and used as an antigen source. Monoclonal antibody was incubated witha protein

A-Sepharose suspension (Pharmacia) for 1 h at room

tem-perature, washed three times with phosphate-buffered

sa-line, and incubated with antigen at 4°C overnight. The

antibodywasthenwashed threetimes withRIPAbuffer and

thenwith SDS-PAGE solubilizingbufferat100°Cfor 5 min

beforeelectrophoresis andWesternblotting.

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a) 1 2 3 4 b) 1 2 a) b)

1 2

_-- -120 o _0-0 -120

4

54

FIG. 1. Western blot of a9oacrylamide gel. (a) Reactivity of R220 (anti-HSV-1 VP13/14) with HSV-1 (lane 1), HSV-2 (lane 2), EHV-1 (lane 3), andEHV-4 (lane 4). (b) Reactivity of13A9 (anti-EHV-1gplO) with EHV-1(lane 1) and EHV-4 (lane 2). Molecular weights of reactive species are indicated in thousands.

Antisera. Monoclonal antibody 13A9 (2) reacts with gplO

of EHV-1. R220 is a monospecific polyclonal antiserum raised in rabbits against SDS-PAGE-purified HSV-1 VP14.

Plasmids andcloning of EHV-4 DNA. The plasmid

contain-ing the 9.6-kb BamHI G fragment of EHV-4 (strain 1942) cloned inpUC9 (6) was digested with Sall, and the resulting left-terminal 1.9-kbBamHI-SaiIfragment and the adjoining 0.9-kb Sallsubfragmentof BamHI-G were separately cloned into theSalIsite of the plasmid vector pUC8 (29) to generate plasmids pUC1.9G and pUCO.9G, respectively.

Bacterial

strains. Escherichia coli JM101 was used for all recombinant DNA experiments. Recombinant plasmids were selected on plates containing 100 ,ug of ampicillin per ml.

Isolation of plasmid DNA.Plasmid DNA was isolated from

bacteria bytheboilingmethod(15)for small-scale

miniprep-arationanalysis. Large-scale isolation ofplasmidDNAfrom

bacteriawas carried out by the alkaline lysis method (21);

the DNAwasfurtherpurifiedbybandingoncesium chloride

gradients containing

50% (wt/vol)

cesium chloride and 200

,ug of ethidium bromide per ml. Plasmid DNA was har-vested, and ethidium bromide was removed by multiple

extractionswithisopropanol. The DNAwasdialyzed against

0.lx TE (1 mM Tris-HCl, 0.1 mM EDTA [pH 8.0]) and recovered by ethanol precipitation.

DNAsequencing.The 1.9 kbBamHI-SalI and0.9-kbSall

subfragments of BamHI-G contained in plasmids pUC1.9G

and pUCO.9G, respectively, were sequenced as previously described (25) by using the dideoxy chain termination method (26) and

[C_-35S]dATP

as a label (4). The Klenow

fragmentofE. coli DNA polymerase I was used for chain

elongation at37°C; the temperature was increased to 45°C

whenregions ofDNA prone to secondary structure

forma-tionweresequenced. PlasmidDNA wassequenced by using

pUC-specific sequencing primers, and EHV-4-specific

syn-thetic deoxyoligonucleotide primers were used to generate

FIG. 2. (a) Autoradiography ofa 12% acrylamide gel to show immune precipitation of [35S]methionine-labeled gplO by 13A9. (b) Western blot of material immune precipitated by 13A9 and probed with13A9 (lane 1)andR220(lane2).Molecularweights of reactive speciesareindicated in thousands. Also shown isreactivity of immunoglobulin released from the immune precipitation matrix (4).

sequence data rapidly. Each nucleotide was sequenced at least three times until anyambiguities were totally removed.

Sequence datawere compiled and analyzed with the

Beck-manMicrogenie sequencing programs.

Nucleotidesequence accession number.Theprimary nucle-otide sequence of openreadingframe B6 has been submitted tothe EMBL datalibraryunderaccessionnumber X 17684 B6.

RESULTS

Cross-reactivity of polyclonal antisera specific for HSV-1 tegument proteins. As part of the characterization of the tegumentproteinsofHSV-1, polyclonalantiserawereraised

againstindividual polypeptidesand tested for

cross-reactiv-ity against a range of a-herpesviruses. One antibody in

particular, R220, showedcross-reactivity. R220was raised

against purified VP14 of HSV-1, and the reaction ofthis

antibody with HSV-1, HSV-2, EHV-1, and EHV-4 on a

k2gtll/EHV-1

insert

Q A P R P R A D F A P P S G E E S S

GAATTCCAAGCACCCAGACCTCGCGCGGACTTTGCACCGCCCTCCGGCGAGGAATCATCT EcoRI

S E E E E E E G PAQA P L D E E D Q L AGAGGAAGAGGAGGAAGAGGGTCCCGCCCAAGCTCCGCTGGACGAGGAAGACCAGCTA

1 Y A D Q Y S V G D S S D E N D E E E D

ATGTATGCTGACCAGTACTCTGTAGGGGACTCTAGTGACGAAAACGACGAGGAAGAAGAC

P R L G S D Y P T S A E S

CCCCGTCTAGGATCTGACTATCCCACGTCCGCCGAATCCAGAATTC EcoRI

18 60

38 120

58 180

71 226

FIG. 3. Nucleotide and predicted amino acid sequence of the

Xgtll

insertcontainingtheepitope recognizedbymonoclonal anti-body 13A9. TheEcoRI sitesareindicated.

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2322 WHITTAKER ET AL.

EHV-4 B6 EHV-1 insert

98 Q A P R S R A D F A P P P E E D S S S E

lII I I

I I

I I I l l

lI

I I

1 Q A P R P R A D F A P P S G E E S S S E

118 E E D E E G P S Q A P L D E E D Q L N Y

I I I I I Il Il lI l Il Il l

21 E E E E E G P A Q A P L D E E D Q LM Y

138 A D Q Y S V G N S S D D N E E D Y L Q P

I II I I I I I I I I

41 A D Q Y S V G D S S D E N D E E E D P R

158 E V E - Y P T S A E S

I I I II I

[image:3.612.65.299.95.229.2]

61 L G S D Y P T S A E S

FIG. 4. Amino acid comparision of the EHV-1 Xgtll insert reacting with 13A9 and the homologous region of open reading frame B6 of EHV-4.

Westernblot is shown in Fig. la. For HSV-1acharacteristic doubletof84,000and79,000Mr, correspondingtoVP13 and VP14of HSV-1, was seen, whereas for HSV-2 only a single

protein of 89,000

Mr,

corresponding to VP14, was

detect-able. Of the other herpesviruses tested only EHV-1 and EHV-4were seen to consistently reactwith R220; for each virus a single band was visible (120,000 Mr for EHV-1, 123,000 Mr for EHV-4).

The molecular weights of the EHV-1 and EHV-4

polypep-tides that reacted with R220 were very similar to those

reported for gplO (1), which raised the possibility that gplO was reacting with R220. A monoclonal antibody to EHV-1

gplO,termed 13A9, hasbeen shown to react with a

[3H]glu-cosamine-labeledpolypeptide ofaround 120,000 Mr (2). The

reactivity of13A9 on a Westernblot is shown inFig. lb.This

antibodyreactedwithpolypeptides of 120,000 Mr in EHV-1

and 123,000 Mr in EHV-4, but no reactivity could be

observed for 13A9 when tested against either HSV-1 or

HSV-2(data notshown).

Todetermine whether R220and 13A9werereacting with the same protein, gplO was first immune precipitated from EHV-1-infected cells(Fig. 2a). This material was then eluted from the immune precipitation matrix, subjected to

SDS-PAGE and Western blotting, and then probed with R220. R220reacted withsamepolypeptideimmuneprecipitatedby

13A9(Fig. 2b).

Identification of the open reading frame encoding EHV-4

gplO. AXgtll expression systemhas beenusedto mapthe

geneencoding EHV-1 gplOtoamappositions0.093to0.114 on the EHV-1 genome (2). Toidentify the gene codingfor gplO,theXgtllinsertcontainingtheepitopethatreactswith 13A9 wassequenced (Fig. 3).Nosignificant homologycould

befound when this sequence was

compared

with the

com-plete DNA sequence of HSV-1 (19). However, sequence

data were available for a region of the EHV-4 genome

between 0.067 and 0.122 mapunits (M. Riggio,

unpublished

data); when the EHV-1Agtllinsertwascompared with

this,

aregion of strong homologywasfound. Acomparision ofthe

predicted amino acid sequences of this region is shown in

Fig. 4.

The homologous region in EHV-4 is part (residues 96

through 169)ofanopenreadingframedesignatedB6(Fig. 5),

which encodes a gene product of 872 amino acids that is

predicted to have amolecular massof 97.4 kDa. The DNA

andpredictedaminoacidsequenceofEHV-4 B6areshown

in Fig. 6, and a plot of the hydropathic profile of the

polypeptide is shown in Fig. 7. Features of the B6 gene

product include a large stretch of around 200 amino acids

toward the N terminus whichis bothhydrophilic andhighly

acidic; theregionbetweenresidues 111 and 201 isespecially

charged,containing26glutamicacidresidues. There appear

tobe nohydrophobic regions of thepolypeptide

character-istic ofeitherasignalsequence(24) oramembrane-spanning

domain(10).

Homology of B6 withHSV-1 andVZV.When openreading

frame B6 wascomparedwith the DNA sequencesofHSV-1 (19) andvaricella-zoster virus(VZV) (8), the geneproduct of

B6showed 17%identitywith the HSV-1 UL47 geneproduct

and 27% identity with theVZVgene 11 product. A

compa-risionofthepredicted productsof these three genes is shown in Fig. 8. The hydrophilic N terminus appears to be a

characteristic ofthese genes;it is conservedin all threebut

with noticeable differences. EHV-4 and VZVbothpossessa

glutamic acid-rich region; instead, HSV-1 contains an

argi-nine-rich region, givingthisproteina highly basic N termi-nus.

a . b I , a h ,,q,m, c a f * , i,o a n d I

-b..S-_r S

b

s I on

=-4--

a

C

I 1-9 0.9 " I

I~~~~~~~~~~~~~~~~~ I

I

II

I .% 1

Be

FIG. 5. Map of the EHV-4 genome showing the restriction enzyme sites and DNA fragments used in studying the genome. The genome consistsofauniquelong region and a unique short region flanked by inverted repeats. (a) Location of the BamHI restriction sites in the viral genome. (b)Genomiclocationof theBamHIGfragment and the 1.9-kbBamHI-SaIlfragment and the0.9-kb Sall-Sallfragment sequenced duringthis study. (c) Location of open reading frame B6. The dot represents the 5' end and the arrowhead represents the 3' end of the gene. J. VIROL.

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GGATCCAGTTCCACCGCTTACCGTAGGG;iATTCGTCAGACTGCTGAAACGCTTGCTCTTCCGTCTAACCTCACCCTACAGAGCATGGAAACTGACGTTCTTGACTACTCATCTATTTCAGG 120 D P V P P L T V G I R Q T A E T L A L P S N L T L Q S N E T D V L D Y S S I S G

MGACGAGCTCAACCAGATGTTTGACATTTAATACAATAAAGCACGTTTCCAAACTTAACATAATGGCCGTATTTTCMGTCGATAMGCTGCGTGAATAGAMCTAATGGGGGGAGGTGGGC 240 D E L N Q M F D I

-GTGGTCTGMGGGTGGTGTATGTTTAAATTGGGCCCGGAGGTCTATAGGCAAGTTTTGTTTGCATTCGTGATCTGCTGCAACAAACGACAATTAACTACCAATCTTCAAATATMGCCCATT 360

TAACAGTACAAAACTAGGGGGTATGGCGGTTTTGAAGCTCGTAGCTTGCCTATAAAACTCGCGCGCCTTTGCMMGA^GTGGATGTTGCTATCTAGMGTAGATAGCGGGCGTTTGCMGTC 480 AAAACTGAMGGTTGTACTACAGCGATACGGAAGTAGTTAGCATGGACCAACATCACGGMGTTMMCGTGGGGMGCCTATAMGCAGGCCTCGCAGATCAATAGAAACGCGCTCCCATCCAT 600 (ORF B6) M D Q H H G V R G G A P I R R P R R S I E T R S H P 26 TTAGAGCCGCAGGAAATACACAGCGCACATACAGCACGCCAGACTTAGTTATAGAGATGGATTGTCTGGCAGAGCCTCTTCACTTGAACCCGGGGGCCAAGCTCACGATCAAAATGAGA 720 F R A A G N T Q R T Y S T P R L S Y R D G L S G R A S S L E P G G Q A H D Q N E 66

GCTCTACACAAGTACTTCAAATAATCAACCAAGCACCTCATTTTGGGGATATCTAMGAAGAGTTTTTTCAGATGATGCCCCCMMCAGCCACAAGCACCAGGTCTCGCGCTGATTTTG 840 S S T a s T S N N Q P S T S F W G Y L R R V F S D D A P A Q P Q A P R S R A D F 106

CTCCTCCCCCCGAGGAGGACTCATCCAGCGAGGAAGAAGACGAGGAAGGTCCCTCACAAGCTCCGTTGGATGAGGAGGACCAGCTCATGTATGCTGACCAATACTCAGTAGGTAACTCTA 960 A P P P E E D S S S E E E D E E G P S Q A P L D E E D Q L M Y A D Q Y S V G N S 146

GTGATGATAACGAAGAAGACTACCTACAGCCAGAAGT TGAATATCCAACTTCCGCAGAAT CTGGCGAATATCATAACAGTGGGATGTTTGCAGAAGGGAGCCGGAAAGCGAGT CTGAGT 1080 S D D N E E D Y L Q P E V E Y P T S A E S G E Y H N S G M F A E E E P E S E S E 186

CAGACATGGAAAACTAMGAAACGTACGAGGAAAATGATACGGAAGTCATATCAGATGATAGCCATAGACTTACTCGTACGTGGTTGGATAGGTCTATAMGCTTAATGGuACGAMGCACTTG 1 200 S D M E N Y E T Y E E N D T E V I S D D S H R L T R T W L D R S I R L M D D A L 226

CACAGTCTTCTGAAATTTCTAAGGCTATCACTAAATCTACGCGCAG-GTTATACGATAGCCAGTTTACTCCAGGGGGTCGAGGCTACAAACAAACGGAAACCCCCTCCCAGCGTTTGGTTC 1320 A Q S S E I S K A I T K S T R R L Y D S Q F T P G G R G Y K Q T E T P S Q R L V 266

ATCTATCAMGCGCTGGTATGTACGATTCTGACGAAATCGTTATGACAGGGGATTACATGGAGGTTGACGACGACCCAAACAGMGCTTACCAGTCATGGGTGCGCGCTATTCACCACCCGG 1440 H L S R A G M Y D S D E I V N T G D Y M E V D D D P N S A Y Q S W V R A I H H P 306

TTGCCATGAACCCATCATGGGAGAAACAATTTCCAATCACACCATACATCGTTTTCTGCCGACATAGACTATGATATAGAMGAGCTAATCGAAATGAACTTGGCGCGAACACCCCAG 1560 V A M N P S W E E T I S N H T N T S F S A D I D Y D I D E L I E M N L A R T P P 346

TGTTTGAGGGATTGCTAG,ACAGMGCAGACTTTTTTTACAGACTACCCATGCTCTATACATATGCTACTATCACTCAAGACGAGGCCTAMGAAGAGCGGCAGGCATGGTCTAATACACAGG 1 680 V F E G L L D S A D F F Y R L P M L Y T Y A T I T Q D E A Y E E R Q A W S N T Q 386

CGCTGCATGGACACGAACAAAGTTCTTGGCCAGCGCTTGTGAGTGATTACTCTAAGGGGGGGATGTACGTGTCCCCTACTCAGGAACCCCGCGGGATATGGCGACGCGCGCTAAACAAG 1800 A L H G H E Q S S W P A L V S D Y S K G G M Y V S P T O E P R G I W R R A L K 0 426

CAATGGCTCTTCAGCTAAAGCTATGTGTGCTTGGTTTAACAGAATTTGTAACTAAGMGTGAGCTCACACAACACCATTCAGCTGTAACTTTTTTGGTCGACTMGCTCCTTAGAACAGCA 1 920 A M A L Q L K L C V L G L T E F V T K R E L T Q H H S A V T F L V D S L L R T A 466

AAAATTGTTACTTGGCCAGCCGACTTTTAGTATTTGCCTGGGAAAGACGCAGGGAAACTGGTGTACGAMGCCCAGCAG,AGCCCCTCATAGCACTCTCCGGGGTTACGCTTCTCCAGCCGC 2040 K N C Y L A S R L L V F A W E R R R E T G V R R P A E P L I A L S G V T L L Q P 506

TTCCCCCAGAAGTCTCAGAATTACTTGAGC,AGCGTACATTTGATATAGGGTTGCGCACCCCCCAAAGTGGAGTGTTTAGAGCGTTCTTCGGACCGCTTGTGTATTGGGCAGAACTACGCA 2160 L P P E V S E L L E Q R T F D I G L R T P a s G V F R A F F G P L V Y W A E L R 546

GAGCCTTGMGAGACCCAGCTGCCATAAACTGTCGCTATGTTGGATTTCATCTCCAAACATCAGAAATTTATTTATTGGCAMGCGCCCACTCTGCCAGCCCGGGCTACACCAAAGAAGAAC 2280

R A L R D P A A I N C R Y V G F H L Q T S E I Y L L A R A H S A S P G Y T K E E 586

TGGTTGCAATGGAGGCAACGCTCACACTTGGGACCCTCATGTTAGAGGTAGCGCTACAGTGGATACACGTGGCCAGTGCACAGTTACTTAGCGAAAACGATGCACTGAAAGCTTTTAGGC 2400 L V A M E A T L T L G T L M L E V A L Q W I H V A S A Q L L S E N D A L K A f R 626

GTGTGAGTGCGTCTATTCCCCAMGCCCTGGMGCCACTTGGTAGCATACGCCTACACGACGCAGAGTTTGAAGTGCTAAGCAACCCAGATGTGATGGTGGCACGTGATGAAACCGCCCTGA 2520 R V S A S I P H A L A P L G S I R L H D A E F E V L S N P D V M V A R D E T A L 666

GCCAGGCGTTGTTTCTTGGATATTTTTCTGTTAGGACCGCACTAACTGCGTGCATGCGTGACTATGCTAATGAGGTGGATGGGGGATCTAAAGAGACCGTTACTGGTTTGTTTTTGGGCG 2640 S O A L F L G Y F S V R T A L T A C M R D Y A N E V D G G S K E T V T G L F L G 706

TGGGGCTAATTATTCAGCGCCTCGCTGGCCATATGAACTTTTTACTAAACTGTATGGCCGGCGCGGCACTTTATGGCGGTAGCAAAATCGCCATACACTCATTAACTCTGCCCAGATACA 2760 V G L I I 0 R L A G H M N F L L N C M A G A A L Y G G S K I A I H S L T L P R Y 746

GCCTATTGGCGGATGTTATGGCCCCTATGCTTCAGCAGCAGTCTTTGGTCGACTTTTGGCGCGCCAGAGACGACATGTTGGAGGAACTAGAAATAACACCACGCCCTGGACCCCCAACGC 2880 S L L A D V M A P M L Q O O S L V D f W R A R D D M L E E L E I T P R P G P P T 786 AAGGCAAGCGCGTGGTGCTGGAGATGCCTTTGCCCTCGGACGATCTTCCAGCTATGACTCCCAGTGGCCAAGTAAACAATGGCGCCGGTTTGGGGCGCATGGTGGACATGGCCAAACACT 3000 Q G K R V V L E M P L P S D D L P A M T P S G Q V N N G A G L G R M V D M A K H 826

TACAGCACTATAGAGAAACAATTATCGGAGACGATGCCTCTTCCTCTGTAGGTAAACGTGGCTTAATGAAATCTGGTGTGGGCGTACGCCATGCGCTGGAGGCGGAGAAAGTAATAAGAT 3120 L Q H Y R E T I I G'D D A S S S V G K R G L M K S G V G V R H A L E A E K V I R 866

ACTCACCCAAAAGCACTTAATGCTGTTTACGTCCCCGGTATGCTCTCACATTCCGCAAGCACTTTCATGAAACCTCTTCTACTTACCTAGCACCCAACTTGTTTGTACGTCTTCGTAACA 3240

Y S P K S T - 872

ATCTATACATTAACTGAATACAATGGAAGCTAGTGGGTCTGCCTCATGGGCCCGMGTTTCCAAAAACCTAATCGAGMGCCGTGCAGTCAAAGGGTGCCTCTTGCCGACCccAAGCGATGT 3360 M E A S G S A S W A R V S K N L I E R R A V K G C L L P T P S D V 33

TATGGACGCTGCTGTTATGGCCTTAAAGACGAACGA 3397

M4 D A A V M4 A L K D E R 45

TFIG.K6kT..Nucleotide.A- andpredicted aminA.A acdsquece f oen eadng

ram240

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Amino

Acid

Number

FIG. 7. Hydrophobicity plotofopenreading frameB6madewith thealgorithm of Kyte and Doolittle (11) andamovingwindowofsix residues. The hydrophilic region (I -)and theglutamic acid-rich region (I-glu 1) areindicated. Positivehydrophobic indices indicate above-average hydrophobicity, and negative indicesindicateabove-average hydrophilicity.

DISCUSSION

The results reported herein indicate that the tegument protein VP13/14 and the glycoprotein gplO of EHV-1 and EHV-4 show both immunologicalcross-reactivity and

pro-teinsequence homology. gplOs of EHV-1 and EHV-4 have been considered major structural glycoproteins, based on

incorporation of [3H]glucosamine (1); EHV-1 gplO has also been labeled by various in vitro labeling methods with

UDP-[14C]

galactoseand [3H]borohydride (28).Thisprotein

wastherefore assumedtobeatypicalherpesvirus membrane protein; i.e., it contains bothahydrophobic signal sequence

atthe Nterminus(24)andasingle hydrophobic

membrane-spanning domain toward the C terminus (10). It is clear from theamino acid sequence ofgplO for EHV-4 that neither of these criteria is fulfilled, which would indicatethat gplO is not present as a typical integral membrane protein. Since

gplO shows both immunological and protein sequence

ho-mology with the tegument protein VP13/14 of HSV-1, it would seemlikely that gplO is presentwithin the tegument region of EHV-1 and EHV-4.

The presence ofa glycosylated protein within the tegu-ment is unexpected, but a protein of this type has been described within the tegument of human cytomegalovirus (3).Thisproteinwasfoundtobeglycosylated by radiolabel-ingwith[3H]galactose andgalactosyl transferase, and itwas

thus considered tobe glycosylated in asimilar fashion toa

number ofcytoplasmicand nuclearproteins that have been found in mammalian systems (13). These proteinsare

glyc-osylatedwithin thecytoplasmof the cellby specific glycosyl transferases,which addshortchainsof N-acetylglucosamine

as 0 linkages. Examples of such proteins include Spl, the

human RNApolymeraseIItranscription factor (12), nuclear

pore proteins suchasp62 (7), and erythrocyte band 4.1 (7,

16). The function of the carbohydrate isnotclear, but itmay

play a role in either nuclear localization or the biological

activity of the protein. Since gplO contains no signal

se-quenceformembrane insertion, it is likelythat glycosylation

occursbyasimilar mechanism, i.e.,additionof

O-glycosyl-linkedN-acetylglucosamine within the cytoplasm.

The levels ofglycosylation of gplO appear to be higher than those of most characterized nonenvelope glycopro-teins. The predicted molecular weight for the B6 gene

product is around 97,000; since the mature protein in the virusparticle hasanestimated molecularweight of 123,000,

this would suggest that carbohydrate residues represent a

molecularweight of26,000.However,noshiftinmobilityof gplOwasfound when theproteinwastreated with endo-or

exoglycanaseenzymes (datanot shown).

The glycosylation of gplO raises the possibility that

VP13/14 is also glycosylated. If this does occur, the

glyco-sylatedproteinmustbe of very lowabundance, since when

HSV-1-infectedcellswerelabeled invivo withhighlevels of

[14C]glucosamine

and

[3H]glucosamine,

no VP13/14 was detected (14, 27). Labeling ofHSV-1 with glucosamine and

immuneprecipitation with R220 failedto showany

conclu-sive

incorporation

of label (data not shown), but it does appearthat

VP13/14,

along withsomeoftheothertegument

proteins of HSV-1, binds certain lectins, indicating that

carbohydrate ispresent (30),albeit at alowlevel.

The immunological cross-reactivity between the

proteins

VP13/14andgplO combinedwith thehomologybetweenthe

genesUL47 and openreading frameB6allow identification of UL47 as the gene coding for VP13/14. UL47 (or open

readingframe B) has been shown to beanimportantHSV-1

gene; it decreases the a-trans-inducing factor-dependent

induction ofa genes (20). A second gene, UL46(or open

reading frame A), adjacent to UL47, has been shown to

activate thea-trans-inducing factor,which in itself is present inthe sameclusterof genes and iscodedforbyUL48. It is

possiblethat thefunction of VP13/14 is sharedbygplO,since

openreadingframe B6 is present inaclusterofopenreading

frames that share homology with HSV-1 UL46, UL47,

UL48,and UL49(24a).The geneclusterfrom UL46toUL49

maps to a position between 0.65 and 0.7 map units on the

HSV-1 genome. The EHV ULregionis inverted relativeto

the HSV UL region (9), so this gene cluster is a position

colinearwith theregion ofEHV-4thatincludesopen

reading

frame B6, apositionbetween 0.05 and 0.15 map units.

It is interesting to note that one of the nuclear and

cytoplasmic glycoproteins currently identified (Spl)

acti-vatesviral and cellular genes. The carbohydrate residueson

Splarethoughttoplayarole in themechanismorregulation

oftranscriptional control, and alleightRNApolymerase II

transcription factors tested from different species were

found to be glycosylated to various degrees (12). It is

possible, therefore,thatglycosylationrepresentsa common

feature ofproteinsthatregulategeneexpression.

J. VIROL.

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EWV-4966

NSV-1UL47 VZVGerm 11

I O) H(] YR GG A PIR9PE SIR T ESs JN F-R A A NT K]E

36 L AVFRSsOITE IIL- ---EIA EETj E[t ~ V RRRR

37 W~T ECJP O L NlE.ATWL V- VS EjLWNSE...J S DAOAWS

72 - TDNNSPrSTSPFW LI.LRRV FJ1- JD PAS0PG0APRSER PAP

67 E0P:NA -. RERERAD -APPTSNEr ASEM-SEP GPOD AA SaS

109 P PRE E J- SI...jiEEjJ R OP SiT PL(f]E ErA] L N YTAOS YT V N S S

102 V 9 0 9 L 0 0 O EI V P 9 G P PR--A 0 G TYI G P V 0 9 A L 0G V G 0 113 919 TCEA D D E gWA ERG j E A IT]D EOGE ~EEOG9 A 990 A

1US SRVOEN0 TPJ FlP PTELJ SYNEyPARE-2.JE NEf G -G iSPlPlK

160RARE Al 00909~~~~~A V[~~] P~~~~S~JROPPEE

186 SORENTr TYTIII... . TOy 1000099IS L TR TWLDER I 9RLEOD 199

7A

0L

O[]PGFP

9DA

WLLLA.P..JVRO

AV10090

A -990 V S- V

CDM

224 ALA0SSREID KAW] EDrST 9t Yfi 00 PQT PSGG 0GYIL0 T1rE TP[

213 LA NPA GTFY jCP OSA F - G .Li : ....-: D VJVW8 PF lDP

229 E~JOVYFTLO EIDWS rST- Oj-EJAL.--- v. JPIm vO

263 SR L[ V N L S i A ONT 0091DE V N TOOTY--NE V 00 P NOV55a W V

243 0DR AV1PFF -E --.S[7TT SafER-A[F jWTIT~ JILDL D0TI91 259 KE (El VSKE CVE9P VTL...-TOO-EIL A--NEN F ESWFP E CT

301 [JA INENPCD- A1N N1PSWE991 ISfi NON TOPSA801010109 E

3.60 N11 R T P P V LOS 0 P9G] L PEL YT YTTAT T1 009 ATYE E90 317 R .jJ - .EL SiSa FJ L TAL P-VGA A

A]M

VP L SASSGl-V

335 T .~ G I ED A -I I R E V 0E

380 SOjWfi N[Do A L NOMN E 1 1PALV K G V OPTS 9 P ljG

369 OPoja G[L -ODAAV Fil LDLGO L--L WIl PI-O--... A.LZLVRA

DL-367 itEoWK -NEK [oJP jWi]VYVAL --PTN A K V NSIE POGF ..v 620 WERF L ~A ~L S TKLCV GEOMRfVTKERR TSG] SA V T

381 R VAAlj!AS NYET AT AL LA RWN POA V C VLTRRE A AIP

658 IivjolL[0 TAKE l T LAO 23 L i V 3A WE9RRRETrv1 R E~ PL;A

618 ILGTV DOVL VENARE T VO0W[~S V-- - -JV-1.-.-..-REIi NPf

"O LLL!AW I0fISArrTJIFLGO N 0N -~- - - --- L V DJI.JW EDRO~

698 ~ ~VTN LS L- FPVSA A ROIE T A--P I LE P 0 V GA

537 v AR -9 L DR ]AnAIN.-C~Ty GPFnLSTSE T Ii-L

678 GjLO~] jRjL IIAN GLSIN L~VflGAA.. yj A- LUNTIA[ L.VT KLEY

506 A~A l T .I~-¶ IiE[0S TOJ- - V yAT GNE A WF -F

573 A [G -L N A . ~L V

517 AGACRC O j RE 0DAAAjT~[I A G L aL Ej FOjA D

562 LKJ--TPSPFlPTE REP LA SF LVGVTGLOOSE-

--605 j EVA aS-N9908 EK FPR SAOEl PEAL APLGOfIR

552 T FDGGFTGTYT 198

586 TO, A9A T T PAK9 P-WAD0V-9RA E9EVOLAP V

6164 Cj~jcD VLLEKISG9V L0IPTWCOOD T T IOjN E PT V TWA

683 A C[RItlTA E V DSG S KEET V 10 10FP V GLAGN 623 SGOTAjp PFALL Di1jf

721 - - NTE CR80 A ALTOSG SEI 8 9SILT IiPR923 L1 0D V NAMI1L

663 RELIEGORE A8AL VS5 99ATfM-10OPPR P (TERN)

691 LLL AALY3PR9~JI D0ArN T[OE 1880A WIT

758 55JJolVDO EAJT ELEREREHTPRP- -GS PT KE

VV---728 ANIJTL TOILJ D 5 A ROTIjbG P110T VI KERV 080 P

793 L N'i P IPSO 0 P A N TP00G VERSGA 0109EV ONE4 ELSEN

768 1[L~ N P R P V 0 9 T T[PJR EL5 P -- -- V: 0 1 E Eki ES 833 1TJI 0D A Sll S G E 0 NEG S1 G V S V R N A I E A E E V I R Y 0 P E S T 802 ELbL[~ ~WEK V A N - LNI Yjt~ (TERN)

FIG. 8. Amino acid comparison between open reading frame B6, HSV-1 UL47, and VZV gene 11. Identical amino acids areboxed.

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ACKNOWLEDGMENTS

This work was supported by grants from the Equine Virology Research Foundation and the WellcomeTrust.

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