JOURNAL OF VIROLOGY,Sept. 1994,p. 5351-5364 Vol.68,No.9 0022-538X/94/$04.00+0
Copyright ©1994, American
Society
forMicrobiologyIdentification 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 71130Received 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 gGandgD 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 5351on November 9, 2019 by guest
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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 withPi
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-proximately25p.g
ofproteinwas loadedperlane. Proteinwasquantitated 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 cellswererehydrated
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 1mgofp-phenylenedi-amineperml) wasoverlaidonthe cells toreduce
fading
(26)
and the cellswere mounted on a glass
coverslip.
Fluorescentcells were visualized
by
using
a model BH2-RFLOlympus
microscope
with anepiscopic-fluorescence
attachment.Purification of EHV-1 virions and
nucleocapsids.
Purified virionswereprepared
fromsupernatantsofL-Mcellscollected at 72 hp.i.
as describedpreviously
(39).
Virions banded in dextran-10gradients
were collectedby
centrifugation,
washed in 1x Tris-EDTA(TE)
buffer,
andresuspended
in I x TE buffer. Viralprotein
wasquantitated
by
using
the BCAprotein
reagent. The
envelope
was removed frompurified
virionsby
detergent
treatment(1.0%
TritonX-100)
for 15 minat37°C
followedby
separation
of theenvelope
andcapsid
proteins by
centrifugation
in a TLA 100.3 rotor in anOptima
TLAultracentrifuge
for30 minat50,000
rpm(54).
Nucleocapsids
were
purified
from the nuclei of infected cells harvestedat 20 hp.i.
as described in detailby
Perdue et al.(37,
39).
Purifiednucleocapsids
wereresuspended
in 1x TEbuffer,
and totalprotein
wasquantitated
by
using
the BCAprotein
assay reagent.Transfections and CAT assays. Transfections of L-M cells and assays for CATwere
performed by
procedures
describedin detail elsewhere
(43,
45,
46).
Plasmids used included anEHV-1 IE
expression
construct(pSVIE),
an EHV-1 IEpro-moter-CAT reporter construct
(IE-CAT),
an EHV-1ICP27
(UL3
gene)
expression
construct(46,
55),
representative
EHV-1early
(thymidine kinase;
pTK-CAT)
and late(HSV-1
USIOhomolog;
pIR5-CAT)
promoter-CAT
constructs(45,
46),
andpSVIR6
(described
above).
Laser-scanning
confocalmicroscopy.
Infectedequine
NBL-6 cells werefixed andfluoresceinisothiocyanate
(FITC)
stained with anti-IR6polyclonal
antiserum as described above. Cells wereexaminedinalaser-scanning
confocalmicroscope
system(no. MRC600;
Bio-RadLaboratories)
attached to a NikonDiaphlot microscope.
Samples
were illuminated with the 488-nmline ofakrypton-argon
laser.Images
through
the cell were taken at1-p.m
intervals with a 60Xobjective
(Nikon
60/1.3).
Individual sections were Kalmanaveraged
over fourscans and stored on a computer.
Images
wereprojected by
using
the Bio-Rad Laboratories COMOS software andwerethree-dimensionally
volumerenderedby
using
INDEC Micro-voxel software.Avideoprinter
(Sony
UP-52000MD)
wasusedto
images.
RESULTS
Production of the
TrpE/1R6
fusionprotein.
Toidentify
and characterize the IR6 geneproduct,
we raised aspecific
poly-clonal antiserum to a bacterial fusion
protein
(TrpE/IR6)
produced
fromtheTrpE/IR6
construct,pPP22path
(Fig. 1).
E. coliTBI cellstransformed withpPP22path
were grownunderinducing
conditions(i.e.,
in the absence oftryptophan).
Theoverproduced
fusionprotein
was isolatedby
separation
through
a 12.5% SDS-PAGEgel
followedby
staining
with Coomassie brilliantblue.Figure
1shows theproduction
of theTrpE/IR6
fusionprotein. Normally,
fusionproteins
within this system fractionatetothe insolubleportion
oflysed
cells,
as wasthecasewith
TrpE/IR6
(lane
3).
Lane2containsanadditionalTrpE/EUS1
fusionprotein
asapositive
control;
lane 4shows the insoluble fractionfromcellstransformed withthepATH22
vector alone. Asexpected,
very little of the fusionproteins
partitioned
tothesoluble fractionsasindicatedin lanes 5to7. The 51.6-kDaTrpE/1R6
protein
wasthenseparated
by
SDS-PAGE,
using
single-well
5%stacking-12.5% resolving
gels
to isolateprotein
in amounts sufficient for immunizations. Thegel
fragments
were emulsified inequal
amounts ofcomplete
Freund'sadjuvant
orincomplete
Freund'sadjuvant
andeither usedto immunize rabbits orstored at-70°C
forlateruse.VOL.68, 1994
on November 9, 2019 by guest
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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 translated1R6protein
and ICPs. Todemonstrate that the antiserum raised totheTrpE/
1R6fusionprotein
wasspecific
forthe1R6protein,
weused the antiserum toimmunoprecipitate
theprotein
product
gener-ated from the in vitrotranscription-translation
of thepIR6G3Z
construct(1R6
ORF cloned intopGEM3Z
[see
Materials and
Methods]). Approximately
106CpM Of[35
S]me-thionine-labeled
proteins
wasimmunoprecipitated, separated
in5.0%112.5%SDS-PAGEgels,
andautoradiographed.
Figure
2 shows that the antiserum to the
TrpE/1R6
fusionprotein
immunoprecipitated
the geneproduct
obtainedby
translation ofincreasing
amounts of1R6mRNA(lanes
5 to7)
butfailed to react with either the translationproducts
of adenovirus mRNA(lane
8)
orthe components of thereticulocyte lysate
(lane 9).
Thesefindings
confirmed that the antiserum wasspecific
for the 1R6 geneproduct.
The antiserum
generated against
theTrpE/1R6
fusion pro-teinwasusedtoimmunoprecipitate
1R6from[35S]methionine-labeledICPs isolatedat
early
and late timesp.i. (see
Materials andMethods).
The data indicated that this antiserum reacted with aprotein
specific
to infected cells, because it was notpresentinthe mock-infected cells
(Fig.
2,lane2).
The molec-ularmassof theprotein
wasapproximately
33kDa,which is inagreement with the
predicted
molecular mass of the 1R6protein.
Theprotein
wasdetectedatbothearly
and late timesp.i. (lanes
3 and4,respectively).
Thefinding
that theapparentsizes of the 1R6
protein
produced by
in vitrotranscription-translation of the
pIR6G3Z
construct and of the1R6protein
detected in infected cells were
quite
similar suggests that extensive modification andprocessing
oftheprotein
does not occur. The lower-molecular-massspecies
probably
representspartial
translationproducts
of the1R6protein.
Time course
analysis
of 1R6expression.
TheTrpE/1R6
antiserum was used toimmunoprecipitate
ICPs isolated at various timesp.i.
todetermine whether thesynthesis
of the1R6protein
correlated with thesynthesis
of the 1R6 mRNA(6).
We
performed
a time course experiment in which ICPs radiolabeled for 1- or2-h intervalswith[35S]methionine
were isolated andimmunoprecipitated
with the1R6-specific
anti-serum. Theimmunoprecipitated
products
wereseparated
by
SDS-PAGE and visualizedby
fluorography.
As indicated inFig.
3, these studies confirmed that the 1R6geneproduct
is---a I -c
-ACGGTGT AAAIAATCTGTATCTCTGAAAAGTCTGTGGT ATTGAGCGTTTCAGCITITTTAATAAAAAAhCGTAAACCATATTTTCCGTGGTGT TGGAGTT TTWWGYACACTCCCCTG
J. VIROL.
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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
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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
J. VIROL.
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[image:6.612.100.266.72.265.2] [image:6.612.347.537.75.406.2]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 excesscoldmethionine 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-infectedcell 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 ,':
VOL. 68? 1994
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[image:7.612.386.482.472.637.2]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. Thephoto-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.
8BandC).
By8 hp.i.,theIR6
protein
has formeda structurethatrings
thenucleus as aseries offlattenedplates,forming
acrown-likeorring-like
structure
(Fig.
8D). VOL. 68, 1994on November 9, 2019 by guest
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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
The American Heart Association. REFERENCES
1. Aguis, C. A., H. S. Nagesha, and M. J. Studdert. 1992. Equine herpesvirus5:comparisonswithEHV-2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure. Virology191:176-186.
2. Audonnet, J. C., J. Winslow, G. Allen, and E. Paoletti. 1990.
Equineherpesvirus type-1 unique short fragment encodes glyco-proteins with homology to herpessimplex virustype-IgD,gIand
gE. J. Gen. Virol. 71:2969-2978.
3. Baker, T. S.,W.W. Newcomb,F. P.Booy, J. C.Brown,and A. C. Steven. 1990. Three-dimensional structures of maturable and abortivecapsids of equine herpesvirus 1 from cryoelectron
micros-copy. J. Virol. 64:563-573.
4. Baumann, R. P., J. Staczek, and D. J.O'Callaghan. 1987. Equine
herpesvirus type 1defective-interfering(DI)particle DNA
struc-ture:the central regionof theinvertedrepeatis deletedfromDI DNA.Virology159:137-146.
5. Ben-Ze've,A., R.Abulafia, and S. Bratosin. 1983.Herpes simplex
andprotein transport are associatedwith thecytoskeletal frame-work and the nuclear matrix in infected BSC-1 cells. Virology
129:501-507.
5a.Breeden,C.A., and D. J. O'Callaghan. Unpublisheddata.
6. Breeden, C. A., R. R. Yalamanchili, C. F. Colle, and D. J.
O'Callaghan. 1992. Identificationandtranscriptionalmappingof
genes encoded at theIR/Us junction of equine herpesvirustype1.
Virology 191:649-660.
7. Bryans, J. T., and G. P. Allen. 1989. Herpesviral diseasesofthe
horse, p. 176-229. In G. Wittman (ed.), Herpesviral disease of J. VIROL.
on November 9, 2019 by guest
http://jvi.asm.org/
[image:12.612.94.263.68.242.2]IR6 PROTEIN OF EQUINE HERPESVIRUS 1 5363 cattle, horses and pigs. Kluwer Academic Publishers, Norwell,
Mass.
8. Caughman, G. B., J. Staczek,and D. J.O'Callaghan. 1985.Equine
herpesvirustype1infectedcell polypeptides: evidence for
imme-diate early/early/late regulation of viralgeneexpression.Virology
145:49-61.
9. Colle,C. F., C. C. Flowers, and D.J. O'Callaghan. 1992. Open
reading frames encodingaprotein kinase, homologue of
glycop-rotein gX ofpseudorabies virus, and a novel glycoprotein map
within the unique short segment of equine herpesvirus type 1.
Virology 188:546-557.
10. Elton,D. M.,I.W.Halliburton, R. A.Killington, D. M. Meredith,
and W.A.Bonass.1991. Sequence analysis of the 4.7-kb
BamHI-EcoRI fragment of the equine herpesvirus type-I short unique
region. Gene 101:203-208.
11. Flowers, C. C., E. M. Eastman, and D. J. O'Callaghan. 1991.
Sequence analysis ofaglycoprotein Dgene homolog within the
unique shortsegmentof the EHV-1genome. Virology
180:175-184.
12. Flowers, C. C., andD. J. O'Callaghan. 1992.Equineherpesvirus1
glycoprotein D: mapping of the transcript and a neutralizing
epitope. J. Virol.66:6451-6460.
13. Flowers,C. C.,andD. J. O'Callaghan. 1992. The equine
herpes-virus type-1 (EHV-1) homologueof herpes simplex virus type 1
US9 and thenature ofamajordeletion within the unique short
segment of the EHV-1 KyA strain genome. Virology
190:307-315.
14. Flowers, C. C.,S. Flowers, B. Tarbet, Y. Sheng, Y.Zhao, S. R.
Jennings, and D. J. O'Callaghan. Synthesis and processing of
glycoproteinDofequine herpesvirustype 1.Virology, inpress.
15. Gray,W. L., R. P. Baumann,A.T.Robertson, G. B. Caughman,
D. J. O'Callaghan, andJ. Staczek. 1987. Regulation of equine
herpesvirustype1geneexpression: characterizationofimmediate
early, early, and latetranscription. Virology158:79-87.
16. Gray, W.L., R. P. Baumann, A. T. Robertson, D. J. O'Callaghan,
and J. Staczek 1987. Characterization and mapping of equine herpesvirus type 1 immediate early, early, and late transcripts. VirusRes.8:233-244.
17. Grundy,F. J., R. P. Baumann, and D. J. O'Callaghan. 1989. DNA
sequence and comparative analyses of the equine herpesvirus type-1 immediate earlygene.Virology150:321-332.
18. Harlow,E., andD.Lane. 1988.Antibodies:a laboratorymanual.
ColdSpring Harbor Laboratory, Cold Spring Harbor,N.Y.
19. Harty, R. N., G. B. Caughman, V. R. Holden, and D. J.
O'Callaghan.1993.Characterization of the myristylated
polypep-tideencoded by the ULlgenethat isconserved in thegenomeof
defective interfering particles of equine herpesvirus 1. J. Virol. 67:4122-4132.
20. Harty, R. N., and D. J. O'Callaghan. 1991. Anearly gene maps
within and is 3' coterminal with the immediate-early gene of
equineherpesvirus 1. J. Virol. 65:3829-3838.
21. Harty, R. N., and D. J. O'Callaghan. 1992. Identification and expression of the ULlgeneproduct of equine herpesvirus1.Virus Res.25:105-116.
22. Holden, V. R., R. N. Harty, R. R. Yalamanchili, and D. J.
O'Callaghan. 1992.TheIR3geneof equineherpesvirustype1: a
unique gene regulated by sequences within the intron of the
immediate-earlygene.DNASequence3:143-152.
23. Holden, V. R., R. R. Yalamanchili, R. N. Harty, and D. J.
O'Callaghan. 1992. ICP22 homolog of equine herpesvirus 1:
expression from early and late promoters.J.Virol. 66:664-673.
24. Holden, V. R., R. R. Yalamanchili, R. N. Harty, and D. J.
O'Callaghan. 1992. Identification and characterization of an
equine herpesvirus 1 late gene encoding a potential zinc finger.
Virology 188:704-713.
25. Joachims, M., and D.Etchinson. 1992. Poliovirus infection results
in structural alteration of a microtubule-associated protein. J.
Virol.66:5797-5804.
26. Johnson,G.D., andG.M. deC. Nogueira Aruajo. 1981.Asimple method of reducing the fading of immunofluorescence during microscopy. J.Immunol. Methods 43:349-350.
27. Koerner, T.J.,J. E. Hill, A. M. Myers,and A. Tzagoloff. 1991. High-expressionvectorswithmultiple cloning sites for
construc-tion of trpE fusion genes: pATH vectors. Methods Enzymol. 194:477-490.
28. Lenk,R., and S. Penman. 1979. The cytoskeletal framework and poliovirus metabolism. Cell 16:289-301.
29. Leung-Tack, P., J. C. Audonnet, and M. Riviere. 1994. The complete DNA sequence and the genetic organization of the short unique region (Us) of the bovine herpesvirus type 1 (ST strain). Virology199:409-421.
30. Maniatis, T., E. R. Fritsch, and J. Sambrook 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
31. McNabb, D. S., and R. J. Courtney. 1992. Identification and characterizationofthe herpes simplex virus type 1 virion protein encoded by the UL35 open reading frame. J. Virol. 66:2653-2663. 32. Nagesha,H. S., B. S.Crabb, and M. J.Studdert. 1993. Analysis of thenucleotide sequence of five genes at the left end of the unique short region of the equine herpesvirus 4 genome. Arch. Virol. 128:143-154.
33. Newcombe, W. W., and J. C. Brown. 1991. Structure of the herpes simplex virus capsid: effects of extraction with guanidine hydro-chloride and partial reconstitution of extracted capsids. J. Virol. 65:613-620.
34. Newcombe, W. W., J. C. Brown, F. P. Booy, and A. C. Steven. 1989. Nucleocapsid mass and capsomer protein stoichiometry in equine herpesvirus 1: scanning transmission electron-microscopic study. J. Virol.63:3777-3783.
35. O'Callaghan, D. J., W. P. Cheevers, G. A. Gentry, and C. C. Randall. 1968. Kinetics of cellular and viral DNA synthesis in equine abortion (herpes) virus infection of L-M cells. Virology 36:104-114.
36. O'Callaghan, D. J., and R. N. Harty. 1994. Equineherpesviruses, p.423-429. In R. G. Webster and A. Granoff (ed.),Encyclopedia ofvirology. Saunders Scientific Publications, London.
37. Perdue, M. L., J. C. Cohen, M. C.Kemp, C. C. Randall, and D. J. O'Callaghan. 1975. Characterization of three species of nucleo-capsids of equine herpesvirus type-1 (EHV-1). Virology 64:187-204.
38. Perdue, M. L., J. C. Cohen, M. C.Kemp, C. C. Randall, and D. J. O'Callaghan. 1976. Biochemical studies of the maturation of herpesvirus nucleocapsid species. Virology 74:194-208.
39. Perdue, M. L., M. C. Kemp, C. C.Randall, and D. J.O'Callaghan. 1974.Studies of themolecular anatomy of the L-M cell strain of equine herpes virus type 1: proteins of thenucleocapsid and intact virion. Virology 59:201-216.
40. Pizer, L. I., G. H. Cohen, and R. J. Eisenberg. 1980. Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virusproduction. J. Virol.34:142-153.
41. Robertson, A. T., R. P. Baumann, J. Staczek, and D. J. O'Callaghan. 1988. Molecularcharacterization of the gene prod-ucts ofthe short region of the equid herpesvirus-1 genome, p. 132-139. In D. G. Powell (ed.), Equine infectious diseases V. Proceedings of the FifthInternational Conference. The University Press ofKentucky, Lexington.
42. Robertson, A. T., G. B. Caughman, W. L. Gray, R. P. Baumann, J. Staczek, and D. J. O'Callaghan. 1988. Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate earlymRNA. Virology 166:451-462.
43. Rosenthal, N. 1987. Identification of regulatory elements of cloned geneswithfunctional assays. Methods Enzymol. 152:704-720. 44. Sharpe, A. H., L. B. Chen, and B. N. Fields. 1982. Theinteraction
ofmammalian reoveiruses with the cytoskeleton of monkey kidney CV-1cells. Virology120:399-411.
45. Smith, R. H., G. B. Caughman, and D. J. O'Callaghan. 1992. Characterization of the regulatory functions of the equine herpes-virus 1immediate-earlygeneproduct. J. Virol.66:936-945. 46. Smith, R. H., Y. Zhao, and D. J.O'Callaghan. 1993. The equine
herpesvirus 1 (EHV-1) UL3 gene, anICP27 homolog, is necessary forfull activation of gene expression directed by an EHV-1 late promoter. J. Virol.67:1105-1109.
47. Southern, P. J., and P. Berg. 1982.Transformation of mammalian cells toantibioticresistance with a bacterial gene under control of theSV40 early regionpromoter. J. Mol. Appl. Genet.1:314-327. 48. Sullivan, D. C., G. P. Allen, and D. J.O'Callaghan. 1989.Synthesis VOL.68, 1994
on November 9, 2019 by guest
http://jvi.asm.org/
5364 O'CALLAGHAN ET AL.
and processing of equine herpesvirus type glycoprotein 14. Virology 173:638-646.
49. Sun, Y., and S. M. Brown. 1994. Theopenreading frames 1,2, 71,
and75are nonessential for thereplication of equine herpesvirus
type I invitro.Virology 199:448-452.
50. Telford, E. A. R., M. S. Watson, K. McBride, and A. J. Davison. 1992. The DNA sequence of equine herpesvirus-1. Virology 189:304-316.
51. Vieira, J., and J. Messing. 1982. The pUC plasmids,an M13mp7 derived system for insertion mutagenesis and sequencing with synthetic primers. Gene 19:259-268.
52. White, E., and R. Cipriani. 1989. Specific disruption of intermediate filaments and the nuclear lamina by the 19-kDa product of the
adenovirusE1Boncogene.Proc. Natl. Acad. Sci. USA 86:9886-9890.
53. White,E., and R. Cipriani. 1990.Role of adenovirus El Bproteins
intransformation: altered organization of intermediate filaments in transformed cells thatexpress the 19-kilodalton protein. Mol. Cell. Biol. 10:120-13(1.
54. Yao, F., and R. J. Courtney. 1989. A major transcriptional
regu-latory protein (ICP4) ofherpes simplex virustype1 isassociated
with purified virions. J. Virol.63:3338-3344.
55. Zhao, Y.,V.R.Holden,R. N.Harty,and D.J. O'Callaghan.1992. Identification and transcriptional analyses of the UL3 and UL4
genesof equineherpesvirus 1, homologs of theICP27 and
glyco-protein Kgenesofherpessimplexvirus. J. Virol. 66:5363-5372.
J. VIROL.