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JOURNAL OF VIROLOGY,Sept. 1994,p. 5351-5364 Vol.68,No.9 0022-538X/94/$04.00+0

Copyright ©1994, American

Society

forMicrobiology

Identification and Initial Characterization of the IR6 Protein

of

Equine

Herpesvirus

1

DENNIS J. O'CALLAGHAN,I* CLARENCEF. COLLE

III,'

C.CLAYFLOWERS,' RICHARD H. SMITH,'

JOSEPHN.BENOIT,2ANDCATHERINEA.

BIGGER'

Departmentof Microbiology and

Immunology'

andDepartment of Physiology andBiophysics,2 Louisiana State University Medical Center, Shreveport, Louisiana 71130

Received 31 March1994/Accepted 23 May 1994

TheIR6 geneof equine herpesvirus 1 (EHV-1) is a novel gene thatmaps within each inverted repeat(IR), encodes apotential proteinof 272 amino acids, and isexpressedas a1.2-kbRNA whosesynthesis beginsat very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D. J.O'Callaghan, Virology 191:649-660,1992). ToidentifytheIR6protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to262) oftheIR6 protein. This antiserum immunoprecipitated a 33-kDa protein generatedby in vitrotranslationof mRNAtranscribedfrom a pGEMconstruct (IR6/pGEM-3Z) that contains the entireIR6 open readingframe.Theanti-IR6antibody also recognized an infected-cell protein of approximately 33 kDa that wasexpressed as earlyas 1 to 2 h postinfection and was synthesized throughouttheinfection cycle. A varietyof biochemical analyses includingradiolabeling the IR6proteinwith oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabelingtheIR6 proteinin thepresenceoftunicamycin,and pulse-chaselabelingexperiments indicated that the twopotential sitesfor N-linkedglycosylation werenotused and that theIR6proteindoes not enter the secretorypathway.Toaddressthepossibilitythattheunique IR6geneencodesanovelregulatory protein,we transientlytransfected an IR6expression constructinto L-Mfibroblastsalone or with an immediate-early gene expression construct alongwith a representative EHV-1 immediate-early, early, or late promoter-chloram-phenicol acetyltransferase reporter construct. The results indicated that theIR6 protein does notaffect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate withpurifiedEHV-1virions and nucleocapsids. Lastly,immunofluorescence andlaser-scanning confocal microscopicanalyses revealed that theIR6 proteinisdistributedthroughout the cytoplasmatearlytimespostinfectionand that by 4 to6hitappearsas"dash-shaped" structures thatlocalize totheperinuclear region. Atlatetimes afterinfection(8to12 h),these structuresassemblearound thenucleus, andthree-dimensional image analysesreveal that theIR6proteinforms acrown-likestructurethatsurrounds the nucleus as a perinuclearnetwork

Equineherpesvirus1(EHV-1)isamajor pathogen of horses and aseriouseconomic threat to the equine industry. Of the fiveherpesviruses that infect the horse, EHV-1 is responsible for significant morbidity and, in the case of virus-induced abortion, mortality (1,7, 36). Currently, several vaccines exist topreventthismostseverethreatof EHV-1infection, but they are insufficient for long-term protection and,as such, are not cost-effective. Therefore, aconsiderable amount of investiga-tion has addressed thereplicationof EHV-1 and the function of its geneproductstogenerateinformation that may be useful in the design of bettervaccines, such as deletion mutants of EHV-1that lack genesessential for virulence in the animal. In this regard, the KyA strain of EHV-1 is ofinterest, because

sequencingof its entire shortregion(6,9,11-13,17,22-24)has revealed that it lacks three openreading frames(ORFs)within the unique short segment (Us) that encode glycoproteins gI andgE andapotential10-kDaprotein(the10Kprotein)which are retained in more recent isolates from horses (50). In addition, these studies have revealed that the Sregion of the EHV-1 genomecontainsseveral genes thatare notpresent in

*Correspondingauthor.Mailingaddress: Department of

Microbi-ology and Immunology, Louisiana State University Medical Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932. Phone:(318)675-5750. Fax: (318)675-5764.Electronic mail address: DCALLA@POP3.LSUMC.EDU.

the genomes of severalAlphaherpesvirinae members, such as herpes simplex virus types 1 and 2 (HSV-1 and HSV-2),

varicella-zostervirus,pseudorabiesvirus, bovineherpesvirus1, and Marek's disease virus (29).

Twogenes that map within the Us segment of the EHV-1 genome appeartobe absent in many members of the

Herpes-viridae

family. These genes are the 10K ORF which maps between the glycoprotein D (gD) andUS9 genes (2, 10)and the EUS4 gene which maps between the genes encoding gG

andgD and encodesapotentialglycoproteinthatlacks homol-ogytoanyherpesvirusgeneproduct describedtodate (9,50).

Withineach inverted repeat(IR)of theEHV-1genome, novel EHV-1genesaretheIR2 gene,an earlygenemappingwithin thesingleimmediate-early(IE)gene(20);theIR3 gene, which is expressed as a late 1.0-kb transcript that mayregulate IE geneexpression byanantisense mechanism

(22);

and the IR6 gene (ORF 67 of the EHV-1 Ab4 strain [50]), which is expressedas amajor early1.2-kb mRNA that issynthesizedin large quantities throughout the infection cycle (6). Recently, DNA sequence analysis has revealed that a homolog of the EHV-1 IR6 gene is present within the Us segment of the genome ofEHV-4 (32).

Inthis paper,wedescribe theidentification of the IR6 gene product and present initial studiestocharacterize this

unique

EHV protein. The findings reveal that the IR6

protein

is a 33-kDa polypeptide that is made throughout the infection 5351

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5352 O'CALLAGHAN ET AL.

cycle and exists as aphosphorylated protein that is present in purified EHV-1 virions and nucleocapsids. The protein is not glycosylated and appears not to function as a regulatory protein. Interestingly, the IR6 protein forms large cytoplasmic structures which appear as cytoplasmic filaments 4 h postin-fection and which, by 8 h postinpostin-fection (p.i.), coalesce into a ring-like or crown-like structure that surrounds the nucleus as a perinuclear network.

MATERIALS AND METHODS

Virus and cells. The KyA cell culture strain of EHV-1 adapted to growth in L-M mouse fibroblasts was used. The methods for virus preparation, virus titer determination by plaque assay, and purification of virions and nucleocapsids have been described previously (35, 37, 39).

Generation of viral DNA clones and PCR. All EHV-1 genomic subclones were grown in Escherichia coli DH5aF'

(Bethesda Research Laboratories, Gaithersburg, Md.) as de-scribed previously (6). The pATH22 expression vector clones were grown in E. coli TB1 (27). Construction and cloning of the pATH22 IR6 expression subclone were performed by standard cloning techniques (30). Briefly, clone pCS-4, which contains the complete IR6 ORF, was digested with PstI and PvuII restriction enzymes (Bethesda Research Laboratories) torelease a 387-bp fragment encoding 129 amino acids of the carboxy-terminal half (residues 134 to 262 in the protein

sequence) of IR6 (see Fig. 1). This fragment was subcloned into the unique SmaI andPstI restriction enzyme sites within the pATH22 expression vector (27) such that an in-frame IR6 fusion ORF would be created with the TrpE ORF. Thus, the pATH22IR6 construct contains a TrpE/IR6 fusion ORF in-serted downstream of the inducible promoter of the trpE operon.

pIR6G3Zwascloned by PCRamplification of theIR6 ORF. Two 28-mer oligonucleotides that hybridized to the 5' and 3' termini of the IR6 ORF were generated. A unique BamHI restriction enzyme site was added to the 5' end of

oligonucle-otide 1

(5'-CGCGGGATCCAAAAGGTAGGGGACTCTC-3'). Oligonucleotide2(5'-CGCGGAATTCTGAAACGCTCA

ATACCAC-3') contained a unique EcoRI restriction enzyme site(see Fig.2). Each oligonucleotide contained a 4-bp exten-sion to facilitate restriction enzyme cleavage. Briefly, pCS-4 was digested withSmaI to release the IR6 insert that was gel

purifiedand used for PCRs. PCRs were carried out by using a kit from Perkin-Elmer Cetus as specified by the manufacturer with the modification of the addition of the Perfect Match Polymerase Enhancer (Stratagene, La Jolla, Calif.) at 1 U/,ug

of DNA template to increase the specificity of the primer extension reactions. PCRs were cycled 27 times at 72°C (2 min), 94°C (1 min), and 55°C (3 min), with an initial denatur-ation temperature of 95°C for 5 min and 55°C for 5 min and a final extended temperature of 72°C for 10 min, followed by holding at 4°C. The resulting amplified IR6 sequence was digested withBamHIandEcoRI(Bethesda Research Labora-tories), purified from gel slices by using the Magic Prep PCR kit(Promega, Madison, Wis.), and cloned into BamHI-EcoRI-digested pGEM3Z (Promega).

Plasmid pSVIR6 was generated for this study by subcloning the IR6 ORF from pIR6G3Z intopSV12. PlasmidpSV12 (45,

46)contains the multiple cloning site from pUC12 (51) down-streamof the simian virus 40promoter-enhancer derived from

pSV2neo (47). This allowed for expression of the IR6 gene product under the control of the simian virus 40 early pro-moter and enhancer. Other constructs used in this study include chloramphenicol acetyltransferase (CAT) reporter

constructs driven by representative EHV-1 early (thymidine kinase; pTK-CAT), late (IR5; HSV-1 US10 homolog; pIR5-CAT), and IE (pIE-CAT) promoters and effectorconstructsof the IE(pSVIE) and UL3 (pUL3;pXG47)genes.Allconstructs have been described in detail elsewhere (45, 46).

In vitro transcription and translation. IR6-specific mRNA was transcribed and translated in vitro by methods described previously (20, 21). IR6 mRNA was transcribed from pIR6G3Z by using a kit from Promega as specified by the manufacturer.IR6 mRNA was in vitro translated aspreviously

described by using a rabbit reticulocyte lysate kit (New En-gland Nuclear Corp., Boston, Mass.) in the presence of [35S]methionine (21, 41, 42). Incorporation of canine pancre-atic microsomes into in vitro translation reaction mixtures to assay for cotranslational processing was done as specified by the manufacturer (Promega). Microsomal vesicles were added in increasing amounts to the in vitro translation reaction mixtures.The mixtures were incubated at

30°C

for 60minand then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis through 5.0% stacking/

12.5% separating acrylamide gels. The 3-lactamase mRNA, supplied in the kit, was translated in the presence and absence of microsomal membranes as a control for signal processing of the protein product.

Generation of the TrpE/IR6 fusion protein. The pATH 22IR6 expression vector (pPP22path) containing nucleotides 11918 to 11532 of the IR segment (encoding 129 amino acids of the COOH terminus of the IR6 protein) was used to transform E. coli TB1 cells, and the fusion protein was purified andisolated by the method of Koerner et al. (27) as described by McNabb and Courtney (31) and Harty et al. (19). The TrpE/IR6 fusion protein was separated through 5.0%/12.5% SDS-PAGE gels and visualized by Coomassie brilliant blue (0.25%) staining in water. The 51-kDa protein was excised and stored at -70°C.

Preparation of TrpE/IR6 antiserum. Gel slices containing the TrpE/IR6 protein were emulsified in an equal volume of either complete Freund's adjuvant (Sigma Chemical Co., St. Louis, Mo.) (for primary injections) or incomplete Freund's adjuvant (for booster injections). Two female New Zealand White rabbits (1 kg each) were given injections of 0.5 ml of this emulsion intramuscularly into the hind legs. Preimmune serum was obtained before the injections were started. Injections of thefusion protein were carried out at 3-week intervals by using procedures described elsewhere (18, 19).

Radiolabeling of RK ICPs and preparation of cell extracts. Rabbit kidney (RK) cells (2.5 x 106 cells per 25-cm2 flask) were infected with EHV-1 KyA at a multiplicity of infection (MOI) of 10 PFU per cell in Eagle's minimal essential medium (EMEM) containing penicillin (100U/ml), streptomycin (100

mg/ml),

nonessential amino acids, 2% dialyzed fetal bovine serum, and 1/10 normal concentration of unlabeled methio-nine for [35S]methionine labeling (8). Infected cells were labeled with [35S]methionine (New England Nuclear) at 50 ,uCi/ml from 6 to 8 h p.i. and harvested in RIPA-1.0% SDS buffer (150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 1.0% SDS, 0.5% sodiumdeoxycholate, 1.0% Nonidet P-40) contain-ing 2.5 mM aprotinin, 2.5 mM leupeptin, and 30 mg of phenylmethylsulfonyl fluoride per ml (8, 48). Infected-cell proteins (ICPs) were radiolabeled with

Pi

by the addition of 100 ,uCiof32P (New England Nuclear) per ml at 6 to 8 h p.i. in EMEM minus P043-. The cells were preincubated in phosphate-free medium for 30minbefore being labeled.

Radiolabeling of early ICPs was enhanced by the addition of phosphonoacetic acid (100 ,ug/ml) to block late-gene expres-sion as described by Caughman et al. (8). Mock-infected cell J. VIROL.

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IR6 PROTEIN OF EQUINE HERPESVIRUS 1 5353 proteins and late ICPs (isolated at 8 h p.i.) were preparedfrom

cells not treated with metabolic inhibitors. In experiments in which tunicamycin was used to inhibit N-linked glycosylation, concentrationsof1, 2, 5,and10 p.g of tunicamycinperml were used (40, 48). The infected cells were labeled with 50 ,uCi of [35S]methionine at 4 to 8 h p.i. as described above. For pulse-chase labeling analysis, infected cellswere preincubated for 30 min inEMEMwithoutmethionine beforebeing labeled and then pulsed for 10 minwith 250 ,uCi of [35S]methionine perml at 5 hp.i. After thepulse, the cellswerewashed several times to remove unincorporated isotope and incubated in mediumwith excessunlabeled methionine(EMEM with 1.0% methionine) for the times listed in thefigure legends.

Radiolabeling of L-M infected-cell extracts with [3H]rman-nose and N-glycanase F analyses. EHV-1 infected cells (MOI, 10 PFU per cell) were starved for glucose for 1 h and then labeled with [3H]mannose (50 1LCi/ml). Cells were harvested at 10h p.i. and lysed in RIPA buffer containing aprotinin (50 ,ug/ml), leupeptin (50

p.g/ml),

and phenylmethylsulfonyl fluo-ride (20 pLg/ml). Cell lysates were clarified by high-speed centrifugation and used for immunoprecipitation analyses.

Anti-IR6 serum (1

jil)

or EHV-1 gD-specific antibody 25 LI)

(13) was added to cell lysates corresponding to 10 cell equivalents. Antigen-antibody complexes were absorbed to protein A-Sepharose beads(Repligen, Cambridge,Mass.),and the proteins were eluted by heating to 100°C for 3 min in elution buffer (50 mM Tris-Cl [pH 6.8], 0.5% SDS, 20 mM 3-mercaptoethanol). For digestion with N-glycanase

(PNGase F; Boehringer Mannheim, Indianapolis, Ind.), the immunoprecipitatesweretreated with 300 mU of PNGaseFin elutionbuffercontaining 0.75% NonidetP-40, andthereaction mixtures were incubated at 37°C for 4 h. The digestion

products were analyzed by electrophoresis through 4 to 20% gradient polyacrylamide MiniProtean Ready Gels (Bio-Rad

Laboratories, Hercules, Calif.).

Western blot and immunoprecipitation analyses. Western immunoblot and immunoprecipitation analyses were per-formed as described previously (12, 22, 45). Nitrocellulose blots containingdenatured viral proteinswere incubated with an anti-TrpE/IR6 antiserum at a final antiserum dilution of 1:20,000. Immunoprecipitationswith anti-TrpE/IR6 were car-riedout at afinal antiserum dilution of1:2,600.AllSDS-PAGE gelswere loaded with 106 cpm of [35S]methionine-labeled or

32Pi-labeled

protein per lane. For Western blot analysis, ap-proximately25

p.g

ofproteinwas loadedperlane. Proteinwas

quantitated by using the bicinchoninic acid

(BCA)

protein

assay reagent (PierceChemical Co., Rockford,

Ill.).

Indirect-immunofluorescence analysis. Semiconfluent mono-layers ofRKcellsgrownon glassslidesweremock infectedor infected with EHV-1 KyA at an MOI of 10 PFU per cell. Infected cells were processed for immunofluorescence at hourly intervals. The cells were washed with

phosphate-buff-ered saline(PBS),fixedincoldacetone

(-20°C)

for 5min, and allowedtoairdry.Forstaining,the fixed cellswere

rehydrated

for 10minin PBS and blocked with normalgoatserumfor 30 to45 min ina humidified chamberat roomtemperature.The slides were washed twice in PBS for 10 min each and then incubatedwith theprimaryantibody(anti-TrpE-IR6or preim-mune serum) at a dilution of 1:5,000 for 30 to 45 min. The slides were then washed twice in PBS for 10 min each and stained with afluorescein-conjugated goat anti-rabbit immu-noglobulin G (heavy plus light chains) F(ab')2 fragments

(Pierce). Following twowashes in PBS, a mounting medium

(90%

glyceroland 10%PBScontaining 1mgof

p-phenylenedi-amineperml) wasoverlaidonthe cells toreduce

fading

(26)

and the cellswere mounted on a glass

coverslip.

Fluorescent

cells were visualized

by

using

a model BH2-RFL

Olympus

microscope

with an

episcopic-fluorescence

attachment.

Purification of EHV-1 virions and

nucleocapsids.

Purified virionswere

prepared

fromsupernatantsofL-Mcellscollected at 72 h

p.i.

as described

previously

(39).

Virions banded in dextran-10

gradients

were collected

by

centrifugation,

washed in 1x Tris-EDTA

(TE)

buffer,

and

resuspended

in I x TE buffer. Viral

protein

was

quantitated

by

using

the BCA

protein

reagent. The

envelope

was removed from

purified

virions

by

detergent

treatment

(1.0%

Triton

X-100)

for 15 minat

37°C

followed

by

separation

of the

envelope

and

capsid

proteins by

centrifugation

in a TLA 100.3 rotor in an

Optima

TLA

ultracentrifuge

for30 minat

50,000

rpm

(54).

Nucleocapsids

were

purified

from the nuclei of infected cells harvestedat 20 h

p.i.

as described in detail

by

Perdue et al.

(37,

39).

Purified

nucleocapsids

were

resuspended

in 1x TE

buffer,

and total

protein

was

quantitated

by

using

the BCA

protein

assay reagent.

Transfections and CAT assays. Transfections of L-M cells and assays for CATwere

performed by

procedures

described

in detail elsewhere

(43,

45,

46).

Plasmids used included an

EHV-1 IE

expression

construct

(pSVIE),

an EHV-1 IE

pro-moter-CAT reporter construct

(IE-CAT),

an EHV-1

ICP27

(UL3

gene)

expression

construct

(46,

55),

representative

EHV-1

early

(thymidine kinase;

pTK-CAT)

and late

(HSV-1

USIO

homolog;

pIR5-CAT)

promoter-CAT

constructs

(45,

46),

and

pSVIR6

(described

above).

Laser-scanning

confocal

microscopy.

Infected

equine

NBL-6 cells werefixed andfluorescein

isothiocyanate

(FITC)

stained with anti-IR6

polyclonal

antiserum as described above. Cells wereexaminedina

laser-scanning

confocal

microscope

system

(no. MRC600;

Bio-Rad

Laboratories)

attached to a Nikon

Diaphlot microscope.

Samples

were illuminated with the 488-nmline ofa

krypton-argon

laser.

Images

through

the cell were taken at

1-p.m

intervals with a 60X

objective

(Nikon

60/1.3).

Individual sections were Kalman

averaged

over four

scans and stored on a computer.

Images

were

projected by

using

the Bio-Rad Laboratories COMOS software andwere

three-dimensionally

volumerendered

by

using

INDEC Micro-voxel software.Avideo

printer

(Sony

UP-52000MD)

wasused

to

print

the

images.

RESULTS

Production of the

TrpE/1R6

fusion

protein.

To

identify

and characterize the IR6 gene

product,

we raised a

specific

poly-clonal antiserum to a bacterial fusion

protein

(TrpE/IR6)

produced

fromthe

TrpE/IR6

construct,

pPP22path

(Fig. 1).

E. coliTBI cellstransformed with

pPP22path

were grownunder

inducing

conditions

(i.e.,

in the absence of

tryptophan).

The

overproduced

fusion

protein

was isolated

by

separation

through

a 12.5% SDS-PAGE

gel

followed

by

staining

with Coomassie brilliantblue.

Figure

1shows the

production

of the

TrpE/IR6

fusion

protein. Normally,

fusion

proteins

within this system fractionatetothe insoluble

portion

of

lysed

cells,

as was

thecasewith

TrpE/IR6

(lane

3).

Lane2containsanadditional

TrpE/EUS1

fusion

protein

asa

positive

control;

lane 4shows the insoluble fractionfromcellstransformed withthe

pATH22

vector alone. As

expected,

very little of the fusion

proteins

partitioned

tothesoluble fractionsasindicatedin lanes 5to7. The 51.6-kDa

TrpE/1R6

protein

wasthen

separated

by

SDS-PAGE,

using

single-well

5%

stacking-12.5% resolving

gels

to isolate

protein

in amounts sufficient for immunizations. The

gel

fragments

were emulsified in

equal

amounts of

complete

Freund's

adjuvant

or

incomplete

Freund's

adjuvant

andeither usedto immunize rabbits orstored at

-70°C

forlateruse.

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5354 O'CALLAGHAN ET AL. J itr A

(1) N N 5 0 N N A Ak T A G T E V r I C A L. A I I N N A N P P N L V L. A P T F

w'

(38) A A A A A G G A A N £ S G F E A P I G E R K N L N P G C N L. G A S Y F

(731 N I E N NE Y A K V P T G 1 F P V A P 1 N V P C R A P A E G V V A G E A L S

(118)Y0 A L. P L P K E K R F 1 L a p 6 T fV R L P F L T P tE E LP

11605~~~~~~~~~~~~~~

114 "f A ArrrCATEACJGrAc TTTJJGINAPE) PC-CCCANA

11485

C B

PPP22path

INSOLUBLE SOLUBLE

- C.. 11 x

1

O&

IL

105

698

433

223

trpE/1R651 61

a

01 :..

S." 54?

[image:4.612.164.463.76.409.2]

FIG. 1. Expressionof theTrpE/1R6fusionprotein.(A)DNAsequence of1R6(6) showingthePstI-PvuII(387-bp)sequence that encodes 129 amino acids(boxed sequence)nearthe COOHterminus of the 1R6protein.ThePstl-PvulIfragmentwassubcloned into thepATH22expression vector.(B) Diagramof thepATHconstructcontainingthe1R6insert.aa,amino acids.(C)Control and recombinantplasmids containingthe1R6 insertweregrown in 100-miculturesofE. coliTB1.The insoluble fractionwasprepared (seeMaterials and Methods)andresuspendedin 2ml of PBS. ForSDS-PAGE,10 p.1of eachsampleper lanewaselectrophoresedand theproteinswerevisualizedbyCoomassie brilliant bluestaining. Lanes: 1, molecularweight(MW) markers; 2, positive control forproduction ofafusion protein by usingapATH23-EUS1 clone (pSE23) to produceaTrpE/EUS1fusionprotein (5a);3,TrpE/1R6from thepPP22pathrecombinantconstructencoding129amino acidsof the 1R6protein; 4,pATH22withoutaninsert;5to7,soluble fractions frompSE23,pPP22path,andpATH22minusinsert, respectively, showingthe absence of fusionproteinswithin the soluble fraction.

Immunoprecipitation

of in vitro translated1R6

protein

and ICPs. Todemonstrate that the antiserum raised tothe

TrpE/

1R6fusion

protein

was

specific

forthe1R6

protein,

weused the antiserum to

immunoprecipitate

the

protein

product

gener-ated from the in vitro

transcription-translation

of the

pIR6G3Z

construct

(1R6

ORF cloned into

pGEM3Z

[see

Materials and

Methods]). Approximately

106CpM Of

[35

S]me-thionine-labeled

proteins

was

immunoprecipitated, separated

in5.0%112.5%SDS-PAGE

gels,

and

autoradiographed.

Figure

2 shows that the antiserum to the

TrpE/1R6

fusion

protein

immunoprecipitated

the gene

product

obtained

by

translation of

increasing

amounts of1R6mRNA

(lanes

5 to

7)

butfailed to react with either the translation

products

of adenovirus mRNA

(lane

8)

orthe components of the

reticulocyte lysate

(lane 9).

These

findings

confirmed that the antiserum was

specific

for the 1R6 gene

product.

The antiserum

generated against

the

TrpE/1R6

fusion pro-teinwasusedto

immunoprecipitate

1R6from

[35S]methionine-labeledICPs isolatedat

early

and late times

p.i. (see

Materials and

Methods).

The data indicated that this antiserum reacted with a

protein

specific

to infected cells, because it was not

presentinthe mock-infected cells

(Fig.

2,lane

2).

The molec-ularmassof the

protein

was

approximately

33kDa,which is in

agreement with the

predicted

molecular mass of the 1R6

protein.

The

protein

wasdetectedatboth

early

and late times

p.i. (lanes

3 and4,

respectively).

The

finding

that theapparent

sizes of the 1R6

protein

produced by

in vitro

transcription-translation of the

pIR6G3Z

construct and of the1R6

protein

detected in infected cells were

quite

similar suggests that extensive modification and

processing

ofthe

protein

does not occur. The lower-molecular-mass

species

probably

represents

partial

translation

products

of the1R6

protein.

Time course

analysis

of 1R6

expression.

The

TrpE/1R6

antiserum was used to

immunoprecipitate

ICPs isolated at various times

p.i.

todetermine whether the

synthesis

of the1R6

protein

correlated with the

synthesis

of the 1R6 mRNA

(6).

We

performed

a time course experiment in which ICPs radiolabeled for 1- or2-h intervalswith

[35S]methionine

were isolated and

immunoprecipitated

with the

1R6-specific

anti-serum. The

immunoprecipitated

products

were

separated

by

SDS-PAGE and visualized

by

fluorography.

As indicated in

Fig.

3, these studies confirmed that the 1R6gene

product

is

---a I -c

-ACGGTGT AAAIAATCTGTATCTCTGAAAAGTCTGTGGT ATTGAGCGTTTCAGCITITTTAATAAAAAAhCGTAAACCATATTTTCCGTGGTGT TGGAGTT TTWWGYACACTCCCCTG

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IR6 PROTEIN OF EQUINE HERPESVIRUS 1 5355

I7n) U I A N U I

119695ACICCCG=TCCCTC

(113) 1 S A L P L

11965CVBABAXCATTAC

(156)A D I I T I

IIT GGAreGCereGie

tCMlTX1MMMTMr.CCAGT.LCCTCACCTCrCTTTGTG

A BT EVT * C AL A aIEIAIPPNL V L AP T P

SI EtEA P E B E NK N L FNPP BC P L GS S P E EI C

B YPA P TP A Y P YAPA P CI V P E B V VABE L

PCB KEPTTTCLUDCTAAAATPIC PPTTTPCAGIBITC

P 9 I I K I f T K a L N D e T f Y I L P f L T P E V T T E 0 E I E P P

T

CATAABGBCCBACBCCBCCB.AIBCBABBBACcACCTcCC38.AAMABCATTCBCCAABBYTYcCCcCocTCGqBAAs TCACADATT

I A O A A DA IS A D P I TL P I I A rAA V P PA

B P KE I P t P AE A T V U K L B F I S L I V T I oA fIEA U rI Fm I

C

It

IJ3$xIlE

05.1 K2i 693

433

[image:5.612.158.458.74.487.2]

1 2 3 4 5 6 7 8 9

FIG. 2. Anti-IR6 antiserumspecifically recognizes in vitro translated IR6 polypeptide. (A) The IR6ORFwasamplified by PCR with primers

corresponding totheoligonucleotides indicated above the IR6DNAsequence (bold line). (B)The DNA productwassubcloned into unique

BamHI andEcoRIrestrictionenzymesites in themultiple-cloning region of the pGEM3Z plasmidto generatetherecombinantplasmid pIR6G3Z.

(C)IR6-specific mRNAwastranscribed from pIR6G3Z in vitro by using the SP6promoter and SP6 RNA polymerase, and the mRNAwas

translatedinvitro. The[35S]methionine-labeled protein productswereimmunoprecipitated withthe antiserumtotheTrpE/IR6fusion proteinat

afinaldilution of1:2,600andwereseparated by SDS-PAGE under reducing conditions. Bandswerevisualized following fluorography.Lanes:1, molecularweight (mw) markers; 2, [35S]methionine-labeled proteins from mock-infected cells; 3 and 4, [35S]methionine-labeled ICPs prepared

fromEHV-1-infected cells under early and late conditions,respectively; 5to7,products of in vitrotranslation of 1, 3, and 5 ,ug, respectively, of IR6in vitrotranscribedmRNA; 8, adenovirus mRNA translated in vitro andimmunoprecipitated with the TrpE/IR6 antiserum (negative control);

9,rabbitreticulocytelysateimmunoprecipitated with theTrpE/IR6antiserum(negative control). Experimental detailsaregiveninMaterials and

Methods.

expressed at early times p.i., as is the IR6 mRNA (6). The 33-kDaIR6proteinwasdetected throughoutEHV-1

replica-tion,beginningat1to2hp.i.(Fig. 3,lane3)andcontinuingup

to 12 h(lane 10).TheIR6geneproductwasdetectedat 24 h p.i. by immunofluorescence analysis (data not shown). As expected, the anti-TrpE/IR6 antiserum did not immunopre-cipitateaproteinfrom mock-infectedcellextracts(Fig. 3,lane 2).Previousstudies (8) involvinghybridselection and in vitro translation hadmappeda31.5-kDaICPtotheIR6generegion and had also shownthatthisproteinwasmadeearlyinEHV-1 infection and could bedetected in large amountsthroughout

infection. Thepresentstudiesconfirm thesefindingsandreveal thatthis majorICP is the IR6geneproduct.

TheIR6protein isnotN-linkedglycosylated.Aspreviously reported (6), the IR6ORF contains two consensus N-linked glycosylation sites at amino acid residues 47 and 232 and a

potential signal sequence at residues 28 to 46. To ascertain whether the IR6 polypeptide is modified by the addition of N-linked oligosaccharides, we performed several experiments toexamine theprocessingandpotentialN-linkedglycosylation of thisuniquegeneproduct.TheIR6proteinwas

immunopre-cipitated from [35S]methionone-labeled cell extracts, and the VOL. 68, 1994

A

1244)

12325

(1)

12205

r uD N t AsA T

Orror Ms=

138) A A A A A--eB BC A A A

120S5

(196)w a

11605TGBATC

(21J)L O

ICCAABcnCCI

11485rCerTTAAATCTGTATCCTG TCT TA =TTTTTA TAACTATTTT=TCTrT TTTT T TC L S I A P O P N PI C A * O PI

B

' a A a v I

sT7.

( plRG3Z

33-283

18.I

ccr.cAr.ccr.c=cv.r..c=AA-C-T-M---I-GCGGCGAGGAGGCCCCCAGAGrAYAMTAASCACCTGTTC"=GYTCGGGYGCATGCTCGGGCGCTCCYACTYCA=WTGTC

GCGAAGWAT GAACQ%GMTACTCGMAACATCCCCACGGGCTACTTLLCCG.CWAMCAGMAGGTGCCGTWZ=TVXXGT Cr.%GG=Tr.GTGGCCGGAGMT WY CA=T

GATTCICTAYCCCATCGrJLGCGCTACr.TGATGAAGCTG=tTCWATACCAGTTGCACGJrCACTUAUCG=TCEAVAASTAAATAMAOCTTCATW=

=r.A CACCCCAGAGOV.CCAGOCAGCCCC1- COCACGTCTCTwAwAAA=CMAACACACCCCCGTGWMTTGAGOCCLUOTGiGAt.

I A I e I T 8

Wo*

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5356 O'CALLAGHAN ET AL.

MW M 1-2 2-3 3-4 4-5 5-6 6-8 8-10 10-12 i05.1

ax-R6

PNGaseF

Pt +

200_ 43.3

33 28.3

18.1

!1 n 3 4 7.1(78 4

FIG. 3. Time course synthesis of the IR6 protein. RK cellswere

eithermockinfectedorinfected with EHV-1 KyA(MOI, 10 PFUper

cell) and were radiolabeled with [35S]methionine (50 ,uCi/ml) at

intervals of1or2 h. Following the labeling, celllysateswereprepared

and 106cpmoflabeledproteinwasreacted with the antiserumtothe

TrpE/IR6fusionprotein. Immunoprecipitateswereanalyzed by SDS-PAGEas described in Materials and Methods. Lanes: 1, molecular weight (MW) markers; 2, mock-infected cell proteins; 3 to 7, ICPs labeled at hourly intervals beginning at I to 2 h p.i.; 8 to 10, ICPs

labeled foraninterval of 2 h from 6to8, 8to 10, and 10to 12 hp.i.,

respectively.

immunoprecipitates were treated with PNGase F, which cleaves essentially all forms of N-linked oligosaccharides. Whenimmunoprecipitates containing IR6wereincubated with

PNGase F(Fig. 4A, lane 5)orPNGase F buffer alone (lane 4) or were untreated (lane 3), no detectable alterations in the

electrophoretic mobility of the 33-kDa IR6 polypeptide was

observed. The activity of PGNase F for digestion of

high-mannose or complex oligosaccharides was demonstrated by PNGase F treatment of EHV-1 gD. EHV-1 gD is present in

infected cells as a 55-kDa precursor that possesses high-mannoseoligosaccharidesandas a58-kDamaturepolypeptide thatcontains complexoligosaccharides (14). Following treat-mentwith PNGase F,both glycosylated forms ofgDshift in molecularsize from55 and 58 kDato43 kDa(comparelanes 6 and 7), the size ofthe unmodified gD polypeptide. These data suggestthat IR6does notcontain N-linked oligosaccha-ridesthatcanbedetectedbyashiftinelectrophoretic mobility

following digestionwith PNGase F. To eliminate thepossibility that theIR6 polypeptide contains non-PNGase-cleavable oli-gosaccharides, cell lysates from EHV-1 infected cells labeled with[3H]mannosewereusedinimmunoprecipitation analyses with anti-IR6 antibody or anti-gD antibody as a positive

control.TheIR6polypeptidewasnotdetectedby immunopre-cipitationoflysateslabeled with[3H]mannose (Fig. 4B,lane5) but was detected by immunoprecipitation of lysates labeled with [35S]methionine (lane 6). As a positive control, gD was

immunoprecipitated from thesameinfected-cell extractsused for analysis of the IR6protein, and the 55-kDagDprecursor

radiolabeledwith [3H]mannose wasreadily detected (lane 1). Both theprecursorandproduct gDproteinsaredetectedwhen gD isimmunoprecipitated from cell lysateslabeled with [35S] methionine.The complete profile is shown in lane 3.

IR6 mRNA was translated in vitro in the presence of

68-

43-

29-loR

... ..A

-3

...~~~~~..r

0.-B_

23s 4... .6 7

B

3H-MAN 35S-MET

(x-g3 PIl(x-gD]

-200

** __-97

_ 58

of -555

55- -43

-29

-18

1 2 3 4

3H-MAN 35S-MET

a-IR617x-IR61

33

5 6

FIG. 4. The IR6 protein does not possess detectable levels of

N-linkedoligosaccharides. (A) The migration of the IR6 polypeptide is notaffected by PNGase Ftreatment. TheIR6 proteinwas immuno-precipitated from infected-cell lysates radiolabeled with [35S]methi-onine (100 uCi/ml) by using anti-IR6 antibody (lanes 3 to 5) or

preimmune serum asthe control (lane 2). Aliquots of the immuno-precipitatewereincubated for 4 h in PNGase F bufferonly (lane 4)or

buffercontaining 300 mU of PNGase(lane 5)or wereuntreated(lane

3), and the digestion productswere analyzed by PAGE in 4 to 20%

gradient gels. The migration of the 33-kDa IR6polypeptide (lane 5)

was notaltered aftertreatmentwithPNGase F. EHV-1 gD immuno-precipitated from thesamecell lysatesby using anti-gD antibodywas

incubated with PNGase F buffer(lane 6)orwith buffercontaining 300 mU of PNGase F(lane 7). Theprecursorandmatureforms of EHV-1 gD migrateas55- and 58-kDaspecies, respectively (lane 6); following PNGase Ftreatment,gD migrates as a43-kDapolypeptide (lane 7). Higher-molecular-mass background bandswereduetolargequantities of protein added. (B) The IR6 protein is not radiolabeled with [3H]mannose. EHV-1-infected cellswereradiolabeledwith

[3H]man-nose (3H-MAN) (4 to 10 h p.i.), and cell lysates were used for immunoprecipitation with either anti-EHV-1 gD antibody (lane 1), preimmuneserum(lane 2),oranti-IR6antibody (lane 5).Celllysates labeled with [35S]methionine (35S-MET) wereused for immunopre-cipitations withanti-gD antibody (lane 3)oranti-IR6antibody (lane

6). The immunoprecipitates were resolved by SDS-PAGE, and the radiolabeledproteins were detected by autoradiographic analysis of thedriedgels. Molecular weightstandardsareshown in lane 4. Details

aregiven in Materialsand Methods.

increasing amounts of canine pancreatic microsomal

mem-branes (see Materials and Methods) to determine possible signalsequenceprocessing.Asindicated in the left-handpanel of Fig. 5a, the migration of the IR6 gene product did not

68.9

A

ax-gD

PNGase F

1-

+1

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IR6 PROTEIN OF EQUINE HERPESVIRUS 1 5357

11(6

/1Ilicrosonels 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1

B-lactaInase

0 0.3 0.6 0.9 1.2 1.5 1.8 2.1

prcL.rsor

,aAmts... `Se.1..W' pr) dsscd

a)

illIIflallc.illVcl l NI 0 2 5 10 min. chase M 0 10' 20' 30' 40' 50'

IR6 _4i0_"_.-,

[image:7.612.125.481.69.255.2]

h) C)

FIG. 5. Analysis of potential signalsequence processing and N-linked glycosylation of the IR6gene product. (a) IR6mRNA, obtained from

in vitro transcription ofpIR6G3Z (Fig. 2), and f3-lactamasemRNA weretranslated in vitro inthe presence of[35S]methionine and increasing amountsofcanine pancreatic microsomes. Proteinproducts were analyzed by SDS-PAGE (5.0 to 12.5% gels) underreducing conditions. (b)

Mock-infected and EHV-1-infected RKcells(MOI, 10 PFU per cell)maintained in the presence of the indicated concentration of tunicamycin wereradiolabeled with [35S]methionineat 4to 8 h p.i. and were harvested at 8 hp.i.Proteins in the lysates were immunoprecipitated (106cpm perreaction) by using the antiserum tothe TrpE/IR6 fusion protein. Immunoprecipitates were analyzed by SDS-PAGE. M, mock-infected cells;

0,untreated,infectedcells: 1,2, 5, and 10, amounts of tunicamycin (micrograms per milliliter) added 1 h prior to infection. (c) Pulse-chase analysis

oftheIR6protein. ICPswereradiolabeled with 250 ,uCiof

[35S]methionine

per ml at 5 h p.i. for 10minand then subjected to a chase with excess

coldmethionine for10,20,30, 40, and50minpostlabeling. The ICPs (10" cpm per reaction) were immunoprecipitated with the antiserum to the TrpE/1R6fusion protein. Experimental detailsfor all three analyses are given in Methods and Materials.

changeinthe presence ofincreasingamountsofmicrosomesin contrast to the f-lactamase gene product, a positive control, which undergoes signal peptidecleavage to a processed lower-molecular-weight mature species. These data indicated that the IR6protein doesnotundergo signalpeptide processing in vitro. Toconfirm furtherthat the IR6protein isnot N-linked glycosylated,weinfectedRK cellswithEHV-1 KyA at anMOI of10PFU percell in thepresenceofincreasing concentrations oftunicamycin, aninhibitorofN-linkedglycosylation whichis useful in identifying precursor-product relationships in the processingof viralglycoproteins (48).ICPs from untreated and tunicamycin-treated cells were immunoprecipitated with the anti-TrpE/IR6 antiserum, analyzed by fractionation throughan SDS-PAGE gel, and visualized by fluorography. As shown (Fig. 5b), the migration ofthe1R6proteindidnotchangewith increasing concentrations of tunicamycin, further indicating that the IR6 proteinis notN-linkedglycosylated.

Lastly,RKcells infectedwithEHV-1 KyA(10PFU percell) weresubjected topulse-chaseanalysis. Priortolabelingat5 h p.i., the infected cells were incubated for 30 min in EMEM without methionine.The infected cells were thenpulsed with

[35S]methionine

at 250 ,uCi/ml for 10 min (Fig. 5c, 0-min chase)andchasedwith an excessofcold methionine(EMEM plus 1.0%methionine) for the times indicated.Asshown(Fig.

5c), the migration of IR6 over time did not change, further

suggestingthat the IR6 geneproductis not N-linked glycosy-lated.Takentogether,these data indicate thatthe IR6protein does not enter the secretorypathway and is not an N-linked glycoprotein.

Phosphorylation of the IR6 protein. Computer analysis of the IR6ORF indicated that theprotein contains sixpotential sites forphosphorylation (6). Therefore,todetermine whether the 1R6 protein is phosphorylated, we labeled EHV-1 KyA-infected RK cells with

32pi

from 1.5 to 8 h p.i.and harvested them atlate timesp.i. (8 h p.i.). TheICPs and mock-infected

cell proteins were immunoprecipitated with anti-TrpE/IR6 serum and analyzed by SDS-PAGE. Following fluorography, theIR6protein (33 kDa)wasobserved in the infected-cell lane (Fig. 6, lane I)andfoundtoberadiolabeled, indicating thatthe IR6proteinis aphosphoprotein.Asexpected, thisprotein was not present within the mock-infected cells (Fig. 6, lane M). Furtherwork isnecessary todetermine whether

phosphoryla-M MW

43.3

:I i. -+-33 28.3

FIG. 6. Phosphorylationof the IR6geneproduct. RKcells,either mock infectedorinfected withEHV-1KyAat anMOI of10 PFU per cell, were radiolabeled with 32Pp (100 ,uCi/mI) at 1.5 h p.i. and harvested at 8 h p.i. Proteins (10' cpm per reaction) from mock-infected (M) orinfected(I) cellswere immunoprecipitated by using

the antiserum to the TrpE/IR6 fusion protein and analyzed by SDS-PAGE andautoradiographyasdescribed in Materials and Meth-ods.MW, Molecularweightmarkers.

a.ENO ,':

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[image:8.612.66.560.80.285.2]

5358 O'CALLAGHAN ET AL.

TABLE 1. Effect ofIR6 expressiononEHV-1 promoter-reporter constructs

CATactivity (%acetylation)'on: Mean CAT activity

Expt' Constructb (%) SD Foldchange

Plate1 Plate2 Plate 3 ()±S

A IE-CAT+pUC-12 20.4 37.7 29.1 +8.7

IE-CAT+pCS-4 5.9 14.7 10.3 ± 4.4 0.4

B TK-CAT +pUC12 0.4 1.3 0.9± 0.5

TK-CAT+pSVIE 24.8 32.4 28.6+3.8 31.8

TK-CAT+pSVIE+pCS-4 21.9 35.0 28.5+6.6 31.6

C IR5-CAT+pUC12 0.1 0.1 0.1±0

IR5-CAT+pSVIE 0.1 1.0 0.6±0.5 6

IR5-CAT+pSVIE+pCS-4 0.1 0.1 0.1±0 0

D IE-CAT+pUC19 28.4 28.4±0

IE-CAT+pSVIR6 23.8 19.0 21.4±2.4 0.8

IE-CAT+pSVIR6 +pSVIE 12.5 10.8 11.7±0.9 0.4

E IR5-CAT+pSVIE 0.7 0.3 0.4 0.5 +0.2

IR5-CAT+pSVIE+pUL3 1.4 0.9 1.5 1.3 ± 0.3 2.6

IRS-CAT+pSVIE+pUL3+pSVIR6 1.4 1.5 1.0 1.3± 0.2 2.6

IR5-CAT+pSVIE+pSVIR6 0.4 0.5 0.4 0.4± 0.1 0.8

aA,B,andC,results obtained with the IR6 gene under the control of its ownpromoter;DandE,resultsobtainedwith the IR6 ORF under the control of the simian virus 40promoter.

bThe EHV-1 reporterandeffectorconstructsandmethodsof transfection and measurement of CATactivityaregivenin Materials and Methods.

c CATactivityresultingfrom EHV-1IE-CAT, early(TK-CAT),andlate(IR5-CAT) promoterswasmeasured as percentacetylationofradioactivechloramphenicol toitsacetylatedderivatives.

tion is mediatedbyavirus-encoded(9, 50)orcellularprotein

kinase.

Effectof IR6 on representativeEHV-1 promoters. Because the IR6 gene product is unique to EHV-1 and appears very

early in the infection cycle, it was of interest to ascertain whether itplays arole in EHV-1 gene regulation. To investi-gate thispossibility, weperformed transient-transfection anal-yses with two IR6 expression constructs and representative EHV-1IE,early, and late promoters linkedtothe CAT gene. Confluent L-M cells (4 x

106

cells per 60-mm dish) were transfected with the various effector and reporter construct combinations by using the Lipofectin reagent (see Materials and Methods). Representative IE, early, and late promoter CAT constructs were pIE-CAT, pTK-CAT, and pIR5-CAT,

respectively(45,46). Thetwo IR6 expression constructs used in these studies included pCS-4 (IR6under the control of its ownpromoter [4]) andpSVIR6(IR6 under the control of the simian virus 40 earlypromoter andenhancer). The results of transactivation assays that examined the possible regulatory function of the IR6 geneproduct are summarized in Table1. Cotransfection of pCS-4 (IR6) with the IE-CAT reporter construct resulted ina slight reduction in CAT activity, prob-ablyas aresult of promotercompetition, because both the IR6 and IE promoters are rich in cis-acting regulatory elements suchasSpI-bindingsites(6, 17). The findingthatthepSV-IR6 constructdidnotsignificantly affectexpression of the IE-CAT constructisconsistent with the above conclusion. Importantly, thepCS-4construct did not alter theability of the IEprotein totransactivate the earlythymidine kinase promoter and did notsubstitute forthe UL3(homolog of HSV-1 ICP27) expres-sionconstruct to mediate expression of the lateIR5 promoter whose expression requires both the IE and UL3 proteins (45,

46).

Likewise, experiments with the pSVIR6 construct showed that the IR6 gene product did not affect expression of the IE promoter and did not prevent the autoregulation of the IE-CAT constructby pSVIE (45, 46). Lastly,experiments with

the pSVIR6 construct confirmed that IR6 expression did not affect transactivation ofa representative late promoter

(IR5-CAT) by the IE and ICP27 (UL3) expression constructs. Although it is possible that the IR6 protein functions in EHV-1 gene regulationby interacting with cellular and/or viral gene products, the results of these transient-expression analyses indicate that the IR6 geneproduct is not a potent transactiva-tor or inhibitransactiva-tor ofexpression of these representative promoter constructs.

Immunofluorescence analysis of IR6 subcellular localiza-tion. A time course study of the cellular location of the IR6 gene product was performed with EHV-1-infected RK cells (Fig. 7). Monolayers of RK cells grown on glass slideswere infected at an MOI of 1 PFU per cell and acetone fixed at hourly intervals (see Materials and Methods). Mock-infected and EHV-1-infected cells were subsequently incubated with eitheranti-TrpE/IR6 antiserum and a goat anti-rabbit immu-noglobulin G(Fab')2 (heavy- plus light-chain) FITC conjugate ornormalrabbit serum plus the FITCconjugate. Controls for backgroundstaining included mock-infected and infected cells fixed at3, 6, and 12 h p.i. and stained with normal rabbit serum plus FITCconjugate orthe FITCconjugate alone. Extensive examination of the cell monolayers revealed that the TrpE/IR6 antiserum reacted specifically with an ICP that appeared initiallyas apunctate pattern throughout the cytoplasm at2to 3 hp.i. (2 h; datanotshown). At 4hp.i., the staining pattern began to coalesce around the nucleus. By 6 h p.i., the IR6 protein appeared as rod-like structures with a perinuclear location. At 8 h p.i., a morepronounced pattern of rod-like structures wasvisible, and by 12 h p.i., IR6 staining exhibited a filamentous pattern throughout the cytoplasm. Further work is necessary to determine whether the IR6 protein associates

specificallywith cellularcytoplasmic proteins and/or elements ofcytoskeletal network as demonstrated with herpes simplex

virus, adenovirus, and poliovirus (5, 25, 28, 52, 53) (see

Discussion). However, preliminary studies with antibodies to

actin, tubulin,andvimentin suggest that theIR6protein does J. VIROL.

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IR6 PROTEIN OF EQUINE HERPESVIRUS 1 5359

[image:9.612.60.557.68.505.2]

I~~ ~ ~ ~ ~ ~ ~ .

FIG. 7. Indirect-immunofluorescence analysis of EHV-1-infected RKcells by using antiserum to the TrpE/IR6 fusion protein. RK cells (approximately 70% confluent),grown onglassslides,wereinfected withEHV-1KyA(MOI, 1 PFU percell)andanalyzedat3 h(A),4h(B), 6h(C), 8 h(D), and 12h(E) p.i. Controls included EHV-1-infectedRKcells stained with normal rabbitserumplustheFITCconjugate (F); infected cells stained with theFITCconjugatealone (G); mock-infected cells stained with normal rabbit serumplus the FITC conjugate (H); mock-infected cells stained with FITC conjugate alone(I); and mock-infected cells stained with anti-TrpE/IR6 antiserum plus the FITC conjugate (J). Mock-infected cellswereharvested at8haftermockinfection. Results obtained by usingacetonefixationareshown; similarresults were obtainedby usingmethanol fixation andparaformaldehyde fixationprocedures.Details are in Materials andMethods.

not specifically interact with any of these cellular structural proteins.

Confocal microscopic examination ofthelocalizationof the IR6protein and formation of perinuclearstructures.Toassess thethree-dimensional distribution of the IR6protein in EHV-1-infected cells, laser-scanning confocal microscopy was used to examine EHV-1-infected equine NBL-6 cells at various timesp.i. Cellswere fixedby acetonetreatment, and the IR6 protein was stained with FITC as the fluorochrome by the same method as for immunofluorescence (see above). For confocalmicroscopy,the infected cellswereopticallysectioned at

1-p.m

intervals at thetimes indicated. The z-axis resolution of theconfocalmicroscopewasless than 0.5 ,um. The

photo-graphs in Fig. 8 represent processed typical images of IR6

fluorescence, each composed of 10 stacked 1-p.m sections takenas ahorizontal slicethroughthe infected cell. At 2 hp.i.,

the IR6 protein appears as small pinpoints distributed

throughoutthecytoplasm (datanotshown).By4hp.i.,theIR6 protein is readily apparent as "dash-shaped" images of fluo-rescence within the cytoplasm (Fig.

8A).

By 6 h p.i., these structuresbecomemoreintense andmorenumerous,exhibita lateral distribution within the cytoplasm, and extend to the ends of the fibroblastpseudopods

(Fig.

8Band

C).

By8 hp.i.,

theIR6

protein

has formeda structurethat

rings

thenucleus as aseries offlattenedplates,

forming

acrown-likeor

ring-like

structure

(Fig.

8D). VOL. 68, 1994

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5360 O'CALLAGHAN ET AL. J. VIROL.

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IR6 PROTEIN OF EQUINE HERPESVIRUS 1 5361 Athree-dimensional rendering of the IR6 crown-like

struc-turethat surrounds the nucleus by 8 h p.i. is shown in Fig. 9A. The photographs in Fig. 9B to D show the IR6 structure presented in Fig. 9A rotated 450, 900, and 1800, respectively. This three-dimensional rendering demonstrates that the struc-ture composed of the IR6 protein rings the nucleus at late times p.i. By 12 h p.i., the cytoplasm of the infected cell has contracted tightly around the nucleus and the crown-like structure composed of the IR6 protein has formed a tight perinuclear network. Examination of a large number of EHV-1-infected cells consistently revealed that the IR6 protein accumulates early in the infected cells as pinpoint structures that seem to aggregate into dash-shaped structures which form the perinuclear crown structure by 8 h p.i. Since the IR6 protein is not encoded by herpesviruses of humans or of animals other than equines and is not related to any cellular protein presently in the database (6), the function(s) of these unusual structuresformed by the IR6 protein in the life cycle ofthe virus is not apparent.

IR6 associationwith virions. To complete the initial char-acterization of the IR6 protein, EHV-1 virions and nucleocap-sids were isolated and purified to determine whether this unique viral gene encodes a structural component of the EHV-1 virion. Purified virions and nucleocapsids were pre-pared by established methods (see Materials and Methods). The virion and nucleocapsid proteins were quantitated by using the BCA protein assay reagent and analyzed by Western blotanalysis with theanti-TrpE/IR6 antiserum. The data (Fig. 10A) demonstrate that the IR6 protein is a component of the virion (lane 1), specifically a component of the nucleocapsid (lane 2). Interestingly, the IR6 protein appears as two species ofapproximately 33 and31 kDa invirions. Virions appearto containapproximately equalamountsof bothforms of theIR6 protein, whereas the larger species is the major form associ-ated with nucleocapsids.

Toexamine further the association of IR6 with viral parti-cles, we treated EHV-1 virions with 1.0% Triton X-100 to separate viral envelope proteins from those of the tegument andnucleocapsid (54). Following centrifugation for 30 min at 50,000 rpm in an Optima TLAultracentrifuge, we subjected

the pellet fraction (nucleocapsid- and tegument-associated

proteins) and the supernatant(envelopeproteins) toWestern blotanalyses with the IR6- andgD-specificantisera (12) (Fig.

7B). The IR6 protein partitioned solely with the pellet fraction, incontrast togD,which, asexpected,wasassociated with the

superuatant

(envelope) fraction and to some extent with the pellet fraction. These data demonstrate that the IR6 gene product is present within the EHV-1 virion andpartitions to thenucleocapsid component.

DISCUSSION

Previous work hasshown thatamajor early transcript of 1.2 kbis expressed in large amountsduringEHV-1 infection(15, 16). This transcriptwasisolatedbyhybrid selectionandshown by in vitro translation to encode a31.5-kDa protein (41, 42), previously identified as a major early ICP during EHV-1 cytolyticinfection (8).DNAsequencing of the genomic locus encoding thistranscript revealed that it is encoded by the sixth gene(IR6; 816 bp) within each of the invertedrepeatsand that the potential protein product of the IR6ORF doesnotexhibit homology to otherherpesviral proteins identified to date (6). The IR6 gene encodes a protein with an as yet unidentified function, although the data presented in this investigation indicate that the proteinmay serve astructural role. Attempts byother workers to generate IR6 deletionmutantshavebeen unsuccessful, suggesting that the IR6 gene (ORF 67 of the EHV-1Ab4 strain) may be essential for virus replication (49). The IR6 protein doesnotcontainmotifs that wouldreadily indicate its membership in a particular functional class of proteins,e.g.,DNA-binding protein, protein withanenzymatic function. Aside from the presence oftwo potential N-linked glycosylation sites andaputative signal sequence, little infor-mation regarding a structure-function relationship can be inferred from theprimary amino acid sequence (6). Therefore, a systematic approachwasundertaken to determine whether IR6encodesaglycoprotein,aregulatory protein,or a nongly-cosylated virion protein. The function of IR6 remains enig-matic; however, the data from these studies indicated the following. (i) The EHV-1 uniqueIR6genedoesnotencode a virion glycoprotein, as evidenced by several experimental approaches to address this possibility. The protein does not undergo processing by N-linked glycosylation into various molecular sizespecies (as is thecase forvirus-encoded glyco-proteins),is notradiolabeledwith[3H]mannose,and does not appearto enterthesecretorypathway. (ii) IR6does notappear toencodearegulatoryprotein.Transient-transfectionanalyses with EHV-1 representative early and late promoter-CAT constructs and the IE expression construct to determine a possible effect of the IR6 protein on CAT expression from these promoters demonstrated that IR6 doesnotparticipateas a regulator of viral gene expression, at least at the level of transcription.IR6 doesnotup-regulateordown-regulateCAT expressionfrom these promoters, does notalter the autoreg-ulation of the IE promoter, andcannotsubstitutefor the UL3 (ICP27 homolog) protein to function in concert with the IE proteintotransactivatealate gene promoter.Itispossiblethat the IR6 geneproductplaysamoresubtle role in EHV-1 gene regulation, but additional studiesarerequiredtoruleoutthis possibility. (iii) The IR6 gene encodes a novel structural

FIG. 8 (UpperPanel). Confocalmicroscopicanalysisof the distribution of theIR6proteininEHV-1-infectedequineNBL-6cells. Equine NBL-6cells infected with EHV-1 KyA strainat anMOIof 1 PFUpercellwere acetonefixedatthe indicated timesp.i.and stainedwithanti-IR6 antibodyat adilutionof1:5,000.Thewedgein thecenterrepresentspixelintensity (brightness)rangingfromnone(0)tomaximalintensity (255). Detailsareexplained in Materials and Methods.(A)Distribution of theIR6proteinin thecytoplasmat4 hp.i. (BandC)Accumulationof the IR6proteinasdash-shaped structuresin both the perinuclear region and cytoplasm at 6 hp.i. (D) By8 hp.i., the IR6proteinappearsas a

perinuclearnetworkthat surrounds the nucleus. The IR6proteindistributes inaring-likeorcrown-likestructure that surrounds the nucleus. FIG. 9 (LowerPanels).Three-dimensional volumerenderingof the distribution of the IR6proteinat8 hp.i. byconfocalmicroscopy.The 10 1-p.m sections thatcompose theimageinFig.8Dwere usedtogenerate athree-dimensional volume rendering of the distribution of theIR6 proteinand todefine the crown-like network of the IR6protein that surrounds the nucleusby8 hp.i. Thedetails of volumerendering and

computeranalysisof the data aregiveninMaterials and Methods.Photographspresentedaretypicalof the distribution andappearanceof the IR6proteinby 8 hp.i.(A)Sameviewas seeninFig.8D.The IR6proteinhas formedacrown-like network that surrounds the nucleus.(B) Image presentedinpanelAisrotated45°.(C) ImageinpanelAis rotated900. (D)ImageinpanelAis rotated1800.

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5362 O'CALLAGHAN ET AL.

A.

anti-IR6

V NC

43.3

28.3

B.

1%TX-100

anti-IR6 nti- gD

S P S

*si ,- es .2.2

1 2 1 2

1 2

FIG. 10. Association oftheIR6 proteinwith purified virions and nucleocapsids. EHV-1 KyAvirions and nucleocapsids were purified

(see Materials and Methods), and viral protein was quantitated by usingthe BCAprotein reagent kit. Samples wereseparated through

12.5% SDS-PAGE gels under reducing conditions and subjected to Western blot analyses. The antibody preparations used included the

antiserum to the TrpE/IR6 fusion protein and an anti-gD peptide

antiserum(12). (A)Purifiedvirions(lane 1)ornucleocapsids (lane 2),

presentat25 p.g perlane. (B) SeparationofEHV-1envelope proteins

from the tegument/nucleocapsid proteins by detergent treatment

(1.0% Triton X-100 [TX-100] for 15 min at 37°C) as described in

Materials andMethods. S, soluble or membrane-associated fraction (lanes 1); P, pellet or tegument/nucleocapsid-associated protein

frac-tion (lanes 2).

proteinthatisreadilydetected inpurifiedvirionsand nucleo-capsids. The data reveal that IR6 partitions to the pellet fraction following detergent treatment of enveloped virions, indicating that it is not associated with the envelope. Since nucleocapsid proteins areconserved among the

alphaherpes-viruses (3, 33, 34, 38), the association of IR6 with EHV-1 nucleocapsidswassurprising. IR6isthereforeprobablyoneof

theminorcomponentsoftheEHV-1 nucleocapsidreported by Perdue et al. (37-39).

Immunofluorescence and laser-scanning confocal micro-scopic analyses to determine the subcellular location of IR6

overtime indicated thattheproteiniswidelydistributed inthe cytoplasm by2 to 4 h afterinfection. By 4 hp.i., the staining patternbeginstolocalizetotheperinuclear region, and by6h p.i., the appearance of filamentous (dash-like) structures

withinthe cytoplasmwasevident.By8hp.i.,these structures aggregateintheperinuclear regionandformadistinct

crown-like structure that surrounds the nucleus in a network-like

arrangement. Since IR6 does not appear tobe a

membrane-associated protein orto be N-linked glycosylated,an

associa-tion with elements of the secretory pathway is notexpected. The staining pattern instead suggested an association with

elements of the infected-cell cytoskeleton. However, experi-mentswith fluorochrome-labeled antibodies toactin, tubulin, and vimentin indicate that these structures composed of the IR6 protein do not colocalize with these cellular structural proteins (datanotshown).

Examplesofviralproteins associated withcytoskeletal

pro-teins have been reported, including an association ofHSV-1

ICP5 (major capsid protein) with the nuclear matrix (5). Additionally,poliovirusassociateswithmicrotubule-associated protein 4 and intermediate filaments ofthecytoskeleton (25,

28). Also, the adenovirusEBB 19-kDa protein associates with the intermediate filament network and allows

anchorage-independent growth for full manifestation ofthe transformed phenotype (52, 53). Lastly, reovirus induces a complete alter-ation of the intermediatefilamentnetwork within infected cells such thatby 12 hp.i.,vimentin filaments,normally

perinuclear,

are reorganized into "wavy" filaments with no apparent orga-nization within the cytoplasm (44). Additional studies are required toascertain whetherspecific cellularproteins interact with the IR6 gene product and whether such interactions, if they occur, play a role in thecytopathic outcome of infection. Thefindings thatthe diploid IR6 gene isperfectlyconserved in the KyA strain of EHV-1 after extensive passage in cell culture (6) and in an EHV-1 wild-type strain (Ab4) isolated from an infected horse in the United Kingdom (50) and that the IR6 gene may be essential for EHV-1 replication in vitro

(49)suggestthat the IR6 geneproduct playsanimportantrole in the life cycle of this virus. The recent report thatORF67 of the EHV-4 genome encodes a potential protein with 65%

homology to the EHV-1 IR6 protein raises the question

whether IR6 homologs may be encoded by EHV-2, EHV-3, and EHV-5. Interestingly, the IR6 homolog of EHV-4 maps within the Us segment and is not present as a diploid gene within the IR sequence as is the case for the EHV-1 IR6 gene. Future work to address the precise function of the IR6 protein and to ascertain definitively whether the IR6 gene is essential for EHV-1 replication in cell culture and in an animal model should yield more insight into the nature of this unique gene product.

ACKNOWLEDGMENTS

We thank Suzanne Zavecz and Scarlett Flowers for excellent technical assistance.

This work was supported by grant Al 22001 from the National

Institutes of Health; a Grayson-Jockey Club Research Foundation, Inc.,research grant; and grants 89-37266-4735 and 92-37204-8040from the U.S. Department of Agriculture Animal Molecular Biology

Pro-gram. J.N.B. issupportedby an Established Investigator Award from

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Figure

FIG.1.vector.produceofaminoinsert4,fusionLanes: pATH22 PBS. Expression of the TrpE/1R6 fusion protein
FIG. 2.BamHIcorrespondingfromtranslatedmolecular9,(C)aIR6 final rabbit Anti-IR6 antiserum specifically recognizes in vitro translated IR6 polypeptide
FIG. 3.TrpE/IR6weighteithercell)andintervalsPAGElabeledlabeled Time course synthesis of the IR6 protein
FIG. 5.wereTrpE/1R6cold0,perofamountsinMock-infected untreated, vitro the Analysis of potential signal sequence processing and N-linked glycosylation of the IR6 gene product
+4

References

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