Vol. 46, No.1 JOURNALOFVIROLOGY,Apr.1983,p.1-14
0022-538X/83/040001-14$02.00/0
CopyrightC1983,American SocietyforMicrobiology
Organization and Expression of the Immediate Early Genes of
Human
Cytomegalovirus
MARK F. STINSKI,l* DARRELL R. THOMSEN,1 RICHARD M. STENBERG,1 AND LYNNC. GOLDSTEIN2
Department of Microbiology, School of Medicine, University of Iowa,IowaCity,Iowa52242,1 and GeneticSystems Corp.,Seattle, Washington 981212
Received8 October1982/Accepted 15 December 1982
The immediate early genes of human cytomegalovirus were characterized
according to map location, RNA transcripts, and translation products. Three
regions in the large uniquecomponent(0.709to0.751mapunits)weretranscribed
in the presence ofaninhibitor of protein synthesis (anisomycin). A single size
class ofpolyadenylated mRNA, 1.95 kilobases (kb), wastranscribed abundantly
relative to the other size classes. The predominant 1.95-kb viral RNA was
transcribed from right to lefton the prototype arrangement of the viralgenome
and spanned a region of approximately 2.8 kb (0.739 to0.751 mapunits). This
mRNA codes fora75,000-dalton protein thatrepresentsthe predominant immedi-ateearly protein detected in infected cells. Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant
immediate early protein is synthesized as a protein of 75,000 daltons, but is
presumably modified in vivo, resulting in abroad banding pattern rangingfrom 75,000to 68,000 daltons. A different immediate early viral gene (0.732to 0.739
mapunits) is transcribed from lefttorightatrelatively low levels. The 3' ends of the
above
viral RNAs terminate at approximately 230 base pairs apart in the region of approximately 0.739mapunits.Five RNA size classes ranging from 2.25to1.10 kbweredetected, but the 1.75-kb and 1.40-kb RNA size classeswere more
abundant from this region. At least four minor proteins are coded by these
mRNAs, withapparentmolecularweights ranging from 56,000to16,500. Last, a
1.95-kb mRNAwas transcribed from a third region (0.709 to 0.728 map units).
This viral mRNA was present at relatively low concentration and coded for a
minorprotein of 68,000 daltons. Since immediate earlygeneexpression of human
cytomegalovirus is dominated by the synthesis ofan mRNAfrom the region of
0.739to0.751 mapunits that codes for the predominantimmediate early protein
found intheinfected cell,wehypothesize that this protein is the major regulatory
protein influencing the switch from restricted toextensive transcription.
The
diseases induced
by
humancytomegalo-virus
(CMV)
frequently
representinfections
af-terreactivation of
latent virus(20, 40).
Reactiva-tion
isbroadly considered
as the event thatallows for
thereplication
of
theviral
DNA and the eventual releaseof infectious
virusto cause overtdisease. Three broad
phases
of CMV
gene expression have been described (6, 12, 15, 46,48, 52, 53):
(i)
theimmediateearly
stage,which
occursin theabsence of denovoprotein
synthe-sis; (ii)
theearly
stage, whichrequires
theaction ofatleastoneimmediateearly
gene; and(iii)
the late stage, which can be detected after viral DNAsynthesis.
The fraction of the genome transcribedincreases
asinfection
progresses(9).
The first viral genesexpressed
afterreactiva-tion
orafterprimary
infection
presumably
codefor
a viralregulatory
protein(s)
that controlssubsequent viral
geneexpression.
These genes arehypothesized
tobe
theimmediate
early
(IE)
viral
genes,which
areexpressed
independently
of
anypreceding
viralprotein
synthesis.
Tran-scription
of the IE viral genes of the Towne(52,
53)
and the Davis(12)
strains of human CMV isrestricted
primarily
toaregion
between
0.66 and 0.77 map units(XbaI-N
and-E)
in thelarge
unique
sectionof
the viral genome. IE viral RNAs thatoriginate
from the aboveregion
areassociated
with thepolyribosomes
aspolyade-nylated
[poly(A)]
RNA of4.8,
2.2,
and 1.9 kilobases(kb) (52, 53).
The IEviral genes repre-sent a restricted andreadily
definable class of viral genes. The viral RNA encodedby
the IE viral genes constitutesapproximately 0.6%
of the infected cell RNA(9).
IEgene
expression
of CMV(Towne)
is char-1on November 10, 2019 by guest
http://jvi.asm.org/
2 ET AL.
acterized
by the presence of an abundant mRNA of approximately 1.9 kb and a predominant protein of 75,000 to 68,000 daltons that is phos-phorylated in vivo (18, 46, 53). The size of the viral protein varies slightly among different strains of CMV (7, 18) and migrates heteroge-neouslyin
denaturing sodium dodecyl sulfate(SDS)-polyacrylamide gels, suggesting
that theprotein
undergoespost-translational
modifica-tions. Other
IEviral
mRNAsand
proteins
arealso
detectable, but they
are presentin
theinfected
cell atrelatively lower concentrations
(46, 47, 52, 53). After
synthesis of
the IEviral
proteins,
thereis
aswitch from restricted
tran-scription
toextensive transcription of
theviral
genome.Synthesis of CMV early
RNAsis
de-pendent
uponthe function of
atleastone IE viral geneproduct. Inhibition of viral protein
synthe-sis with inhibitors
such as cycloheximide (12, 13,52, 53)
orby
treatmentwith interferon
(47) haslittle
to noeffect
on IE RNAtranscription,
butthe
switch from restricted
toextensive
transcrip-tion is inhibited.
Therefore, it is proposed
thatthe
IEproteins of CMV have
animportant
role in controlling viral gene expression.This
reportdescribes
theorganization
of the IE genesof CMV in the region of 0.709 to 0.751 map units(XbaI-E).
The size of the transcripts in the absence of de novo protein synthesis, thedirection of transcription,
and theproteinscod-ed for
by
the IE mRNAs arereported.
The IE geneexpression of this herpesvirus
isdominated
by
thetranscriptionof
asingle
gene that allowsfor the translation
of
apredominant
IEprotein.
Theemphasis of this
reportis
onthis
predomi-nant IE gene.The
adjacent
IE genes areana-lyzed
for the
purposeof comparison. The
tran-scription in the adjacent region of approximately
0.660 to 0.685 mapunits
(XbaI-N)
atvarious
times after infection requires further
investiga-tion.
MATERIALS AND METHODS
Virus and tissue culture. Humanfibroblast cells and the plaquepurification of human CMV (Towne strain) werepreviously described (46). The amount of infec-tious viruswasdeterminedby assays for plaques (57) ortissuecultureinfective doses(17).
Viral infection and definitions of IE.Forisolation of IE RNA, cells treated with 100 ,uM anisomycin 1 h before infection wereinfected with CMV at a multi-plicity of 10 to 20 PFU per cell in the presence of anisomycin. After12hinthepresence ofanisomycin, thecellswereharvested.Atthe concentrations usedin these experiments, anisomycin inhibited protein syn-thesis by 99% orgreater. Forpulse-labeling IE pro-teins,cells were treatedwith200
p.g
ofcycloheximide per ml instead ofanisomycin. After removal of the cycloheximide,infected cells werepulse-labeled with[35S]methionine
for3 hin thepresence ofhighsalt and actinomycin D as previously described(46). In addi-tion, infected cells as well as uninfected cells werepulse-labeled with [35S]methionine for 3 h without treatmentwith cycloheximide oractinomycin D. In-fected cells were pulse-labeled in high salt, whereas uninfected cells werepulse-labeled in normal saltas previouslydescribed (46).
Isolation of RNA. All reagents, plasticware, and glassware were treatedwith0.1% diethyl pyrocarbon-ate (Sigma Chemical Co., St. Louis, Mo.) and auto-clavedbeforeuse. Thccellsweresuspendedin 25 mM Tris-hydrochloridc (pH 7.5)containing 25 mMNaCl,5 mMMgCl2, 2% Triton X-100, 5% RNase-free sucrose, 100 ,ugof heparin -;cr t and 40 Uof RNasin(Biotec, Madison, Wis.) anc' lisrupted withaDounce homoge-nizer (Bpestle). Poi-<-me-associated RNAwas isolat-edby the magnesium ;recipitation method of Palmiter (35) as previously described (52, 53). Poly(A) RNA was selectedfrom total polysome-associatedRNAby oligodeoxythymidylic acid-cellulose chromatography (type 2; Collaborative Research, Inc., Waltham, Mass.)aspreviously described (53).
Physical map of the XbaI-E region. The cloning, purification, and characterization of recombinant plas-mids containing insertions of CMV DNAhave been described previously (51). Recombinant plasmids pCB45 and pCB42were agift from R. La Femina and G.Hayward. The recombinantplasmid containing the XbaI-E insert was mapped by the end-labeling and partial digestion method of Smith and Birnstiel (42) andby double restriction enzymedigestions of various fragments of the XbaI-Eregion. Restriction endonu-cleaseswereobtained from Bethesda Research Labo-ratories, Inc., Rockville, Md. The conditions were as describedby the supplier.DNAfragments were frac-tionatedbyelectrophoresis in1.0 to1.5% agarose gels by the method of Bachi and Arber (3). VariousDNA fragmentswere isolatedfrom agarose gels by electro-elution and ethanolprecipitated.
Preparation of radioactive probes. Recombinant plasmidDNAs werelabeledwith[a-32P]dCTPby nick translationasdescribedby Rigbyetal.(41). Radioac-tive cDNA probes to viral RNA were prepared as follows.Approximately35 ,ugofIEpoly(A)RNA was used for selectivehybridizationtoXbaI-EDNA cova-lently bound todiaminobenzyloxymethyl (DBM)-cel-luloseaspreviously described (53). After elution from the DNA-cellulose, the RNAs were rechromato-graphed on oligodeoxythymidylic acid-cellulose and thenusedastemplates for thesynthesis of 32P-labeled cDNA. Synthesis of cDNAtothecomplete sequence of the mRNA (total cDNA) byreversetranscriptase (avianmyeloblastosis virus)wasprimedwith 2.5,ugof randomoligodeoxynucleotide from calf thymusDNA in 50,ul of reactionmixture.Approximately200ngof viral RNAand12.5 Uofreversetranscriptase in 25 mM Tris (pH 8.3) containing 35 mM KCl, 5 mM MgCl2, 2.0 mM dithiothreitol, 15 p.M each dATP, dGTP, and TTP, 3 p.M dCTP, and 100 ,uCi of
[a-32P]dCTP
(3,000Ci/mmol;AmershamCorp, Arlington Heights, Ill.) was incubated at 37°C for 1 h. The reactionwasstopped by the addition of SDSto0.5% and EDTA to 20 mM. The reaction mixture was treatedwith 25p.gofproteinaseK at37°C for45min and thenphenol-chloroformextracted. RNAtemplate was degraded by incubation at 37°C in 0.3 M NaOH for 16 h. The cDNA was separated from unincorporated nucleotidetriphosphates by gel filtrationonSephadex G-50.J. VIROL.
on November 10, 2019 by guest
http://jvi.asm.org/
IMMEDIATE EARLY GENES OF HUMAN CMV 3
The cDNAtothe 3'end of the RNAs(3' cDNA)was
synthesized as described above, except the RNA templatewasalkalinedegraded in0.1 NNaOH for1h
atroomtemperaturebefore chromatographyon
oligo-deoxythymidylic acid-cellulose and0.33 ,ugof oligo-deoxythymidylic acid (Collaborative Research, Inc., Waltham,Mass.)wasusedasprimer. Theaveragesize
of3' cDNAwasdeterminedtobe 180 nucleotides by methylmercury gel electrophoresis.
Gel electrophoresis of denatured poly(A) RNA. Poly(A)RNAwasfractionatedby electrophoresis ina
1.5% agarose slab gel containing 10 mM methylmer-curyhydroxide (Alpha,Danvers, Mo.)asdescribedby
Bailey and Davidson (4). Approximately 10,ugof IE poly(A)RNAper cm2ofgel slotwasused for electro-phoresisinpreparative slab gels. Electrophoresiswas at 11.5 mA/cm2 at 15°C for4.5 h. Molecular weight standards were 23S (3.3-kb) and 16S (1.7-kb)
Esche-richia colirRNA(5) and28S (5.3-kb) and 18S (2.0-kb) human cellrRNA (32).The standardswerevisualized by stainingin 1,ug ofethidiumbromideperml and 0.5 Mammonium acetate. The sizesoftheviral mRNAs wereinterpolated fromastandardcurve.
Northern blot hybridization. Gels containing IE polysome-associated,poly(A)RNAwerepreparedfor
transfer to DBM-paper (Schleicher & Schuell Co., Keene, N.H.)aspreviously described(53). The
DBM-paper was activated as described by Schleicher &
Schuell Co. The RNA blotswereincubatedat42°C for 24 h in prehybridization buffer containing Sx SSPE (1x SSPE is10mM NaPO4[pH 7.7], 0.18 M NaCl, 1
mM EDTA), 50% formamide, 0.08% (wt/vol) each bovine serum albumin, Ficoll, and
polyvinylpyrrol-idone,1%glycine,0.1%SDS,and 200,ug of denatured calfthymus DNA perml. Radioactive probes
repre-senting various sections of the XbaI-E region and havingaspecific activityof 1 x 108to2x 108 cpm/,ug
were heated to 100°C for 3 min and then added to
hybridization buffer preparedasdescribed above, ex-ceptthebovineserumalbumin, Ficoll, and
polyvinyl-pyrrolidone were 0.04% each. Hybridizations were
withanequal molar quantity ofDNAand
radioactiv-ity, depending on the size of the DNA fragment. Hybridizationwas at42°C for72 h. After hybridiza-tion,the RNA blotswerewashed three times with5x SSPEcontaining0.1%SDSatroomtemperatureand three times with 0.1x SSPEcontaining 0.1% SDSat
50°C. Hybridizationofthe 32P-labeled DNAprobesto
CMV-specific RNAwasdetected by autoradiography with X-Omat AR film (Eastman KodakCo., Roches-ter, N.Y.)
Southern blot hybridization. Recombinant plasmid pMW34 (BamHI-A fragment of the XbaI-E region) (Fig. 1)wasdigestedwithrestrictionenzymesBamHI and PstI (1 U/,ug; Bethesda Research Laboratories). Recombinant plasmid pCB42 (BamHI-B fragment) (Fig. 1)wasdigestedwithrestrictionenzymesBamHI, PstI,andSalI. The DNAfragmentswerefractionated in1.0%agarosegels bythe method of Bachi and Arber
(3)andthen immobilizedontonitrocellulose filters by themethod of Southern(43). Filterscontaining immo-bilized DNA were pretreated at 42°C for 24 h with prehybridization buffer containing 5x SSPE, 50%
formamide, 1.0%eachbovine serumalbumin, Ficoll, andpolyvinylpyrrolidone, and 200,ugof denaturedcalf thymusDNAperml.32P-labeled recombinantplasmid XbaI-E DNA, 32P-labeled total cDNA synthesized
from viral RNA template complementarytoXbaI-E, and32P-labeled 3' cDNA synthesized from the 3' ends of viralRNAtemplatecomplementarytoXbaI-Ewere prepared as describedabove. Approximately 500,000 cpm of each 32P-labeled probe was suspended in water, heated to 100°C for3 min, and then addedto hybridization bufferpreparedasdescribedabove, ex-ceptthe bovineserumalbumin, Ficoll, and polyvinyl-pyrrolidone were 0.04% each. Hybridization was at 42°C for 48 h. Theamountof immobilized recombinant plasmid DNA was at least 50-fold greater than the amount of input 32P-labeled probe. After hybridiza-tion, the filters were washed twice with 2x SSPE containing0.1% SDSat roomtemperature and twice with 0.1x SSPE containing0.1% SDS at50°C. After final washing with 2x SSPE, hybridization of 32p_ labeled probewasdetectedbyautoradiography.
Hybridizationof viral RNA to DNA bound to DBM-paper. Recombinant plasmid DNAs representing the PstI-D, Sacl-A, and SalI-C (Fig. 1) sections of the XbaI-Eregionorthe entireXbaI-Eregionof the viral genomeweredigestedwithrestriction enzymePstI or BamHI, denatured, and covalently linked to DBM-paperby the method of Stark and Williams(44). Each DNApreparation hadapproximately 105 cpm of 32p_ labeled recombinant plasmid for estimating the per-cent DNAlinkagetotheDBM-paper. Generally,40% oftheinputradioactivity remained boundtothe DBM-paper, whichwasextrapolatedtoapproximately15,ug of recombinant plasmid DNA linkage. The linked DNA was at approximately 150-foldexcess for each microgram of RNA hybridized.
The input IE poly(A) RNA was 20 ,ug for PstI-D DNA, 80 ,ug for Sacl-A DNA, and 100p.gfor Sall-C DNA.After thepoly(A)RNA washeatedat80°C for2 min and cooledonice, itwasaddedto ahybridization buffer of 20mMPIPES (piperazine-N,N'-bis(2-ethane-sulfonicacid), pH 6.4,containing0.4 MNaCl,5 mM EDTA, 50% deionizedformamide, 0.2% SDS, and1 ,ug of tRNA from calf liver (Boehringer Mannheim Corp., Indianapolis, Ind.) per ml.Hybridizationwasat 57°C for5 h. TheDBM-paper wasthenwashed three times with 2x SSPE containing 0.2% SDS at room temperatureandsix timeswith0.1x SSPEcontaining 0.2% SDSat60°C for 15-min periods. Thehybridized RNAwaseluted in99% deionized formamide contain-ing 10 mM PIPES (pH 6.4) at 70°C. Under these conditionsapproximately20 to 100ngofvirus-specific RNA wasisolated. TheviralRNAwasethanol precip-itated with10,ugof calf livertRNA ascarrier.
R-loopsand electronmicroscopy.IE polysome-asso-ciated, poly(A)RNA wasselectedby hybridizationto recombinant plasmid XbaI-E DNA bound to DBM-cellulose bythe method ofNoyes and Stark (34) as previously described (1, 53). Approximately50,ugof IEpoly(A)RNA washybridizedtoapproximately50 ,ug of recombinant plasmidXbaI-E that was cleaved bydigestionwith therestriction endonucleaseBamHI andbound tocellulose. Hybridizationwasfor16h at 57°C in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 8.0, containing 70% deionizedformamide,0.5 MNa+,and 0.005 M EDTA. After hybridization, the cellulose was rinsed twice with 2x SSC(lx SSC is 0.15 MNaCl plus 0.015M sodium citrate) at room temperature, twice with hy-bridization buffer at room temperature, and three timeswith hybridization bufferat60°C. The hybrid-VOL.46,1983
on November 10, 2019 by guest
http://jvi.asm.org/
ized RNA was eluted in 99%formamide containing 10 mMHEPES (pH 8.0) at 60°C. The eluent was diluted to afinal formamide concentration of22% and adjust-ed to0.2 M sodium acetate, and then the RNA was ethanol precipitated. Approximately 5% of the input RNAwasrecovered. The selected IE RNA (2 to 3 ,ug) was incubated at 80°C for 2 min, cooled on ice, and thenincubated at 65°C for 10 min with 1 ,ug of XbaI-digested, recombinant plasmid XbaI-E DNA in hy-bridization buffer prepared as described above. The mixture was then incubatedat57°Cfor 16 h. Hybrid-ization of RNA to double-stranded DNA and the formation of R-loops were detected by electron mi-croscopy. The samples were mounted for electron microscopy by theformamide technique, stained with uranyl acetate, and shadowed withplatinum-palladium by the method of Thomaset al.(50). Vector plasmid pACYC 184 (4.2 kb) and M13 DNA (7.2 kb) were used assize standards. R-loops presentin18representative molecules wereanalyzed.
Invitrotranslationof RNA andanalysisof polypep-tideproducts.IEpoly(A)RNAselectedby hybridiza-tion was ethanol precipitated twice in 2% sodium acetate and once in 2% potassiumacetate. The viral RNA was dissolved in a minimal volume ofsterile water,freeze-dried, and suspended in 10
,u1
ofsterile water. Viral RNA was translated in vitro by using a micrococcal nuclease-treated rabbit reticulocyte ly-sateprepared by the method of Pelham and Jackson (37). Each 22-,ul assay mixture contained 10RI1
of reticulocyte lysate, 25 11Ci of[35S]methionine
(1,225 Ci/mmol; Amersham Corp, Arlington Heights, Ill.), and10,ul ofmRNA in water.Afterincubationat37°C for 60min,5,ulof pancreaticRNase(100p.g/ml)
and 20 U ofT,
RNasepermlin25 mMEDTAwereadded, and themixturewasincubated foranadditional 10 min at37°C.Infected cells were pulse-labeled with [35S]methio-nine underIEconditionsaspreviously described (46). Polypeptides from in vitro translations, pulse-la-beled infectedcells, orimmunoprecipitateswere frac-tionated by electrophoresis in discontinuous SDS-polyacrylamide slab gels by the method of Laemmli (26)aspreviously described (46). After the gelswere stained and destained as previously described (45), they were soaked in water, and then 1 M sodium salicylate was added for fluorography (8); the gels were dried and exposed to Kodak X-Omat AR film. Molecularweightsweredetermined from their migra-tion relative to unlabeled molecular weight markers (45, 46) and labeled adenovirus polypeptidesfrom in vitrotranslation (31).
Immunoprecipitation ofvirus-specifiedpolypeptides. Infectedanduninfectedcells werepulse-labeled with
[35S]methionine
as described above. Extracts of [35S]methionine-labeled cells were prepared for im-munoprecipitation as described by Goldstein et al. (19). The extractionbuffercontained10,ug of phenyl-methylsulfonyl fluoride per ml to inhibit proteolytic enzyme activity. Immunoprecipitation of immune complexes by Formalin-fixed Staphylococcus aureus Cowan I strain was by the method ofKessler (25). Appropriate samples of[35S]methionine-labeled pro-teinscontaining 105 cpmwereincubatedat4°Cfor 60 min with a 1:40dilution ofnormal mouse serumandan equal volume ofa10%suspensionof S. aureus. The S. aureuswaspelletedbycentrifugation, and thesuper-natantswereremoved.Monoclonal antibodyE-3(19)
ornormalmouseserum wasaddedtothesupernatants at afinal dilution of 1:100. After 60 minat 4°C, an
equal volume ofa10% suspension of S. aureus was
added, andthe mixture wasincubated at4°C for 60 min. The S.aureus waspelleted by centrifugation, and the pellet was washed five times in 0.1 M Tris-hydrochloride (pH 8.0) containing 0.5 M LiCl and 1.0% beta-mercaptoethanol. The sample was then suspended in dissociating solution for SDS-polyacryl-amide gel electrophoresis (26) and boiled for 3 min, and the S. aureus was pelleted by centrifugation. Bromophenol blue and glycerol were added to the supernatants, and the samples were fractionated by SDS-polyacrylamide gel electrophoresis aspreviously described(45, 46).
Thegelswerestained, destained, and prepared for fluorography as described above. Molecular weight standardswerealsoasdescribed above.
RESULTS
Physical
mapof theXbaI-E
DNA.Abundant
IE
RNA of human CMVoriginates
from the XbaI-N(6.9-kb)
and -E(20.0-kb)
region (0.660 to 0.770 mapunits) of the viral genome (52, 53).Physical
mapsof the XbaI-EDNA were generated by the method ofSmith and Birnstiel (42) and by dou-ble restriction enzyme digestions of various frag-mentsof the XbaI-E region. Figure 1illustrates
the various physical maps of the XbaI-Eregion
and therelationship
of this region to theXbaI
physical map of the entire viral genome deter-minedby La Femina and Hayward (27; submit-ted forpublication). The AvaI, SmaI, and HinclI sitesweremappedby double restrictionenzyme digestion of defined subclones of the XbaI-E region;consequently, these sitesarenotmapped for the entire XbaI-E region. The recombinant plasmid designations for various subclones of the XbaI-Eregion and relevantmapunitsofthe region arealso indicated in Fig. 1.Mapping
andsizedistribution ofIERNA. The IEpolysome-associated poly(A)
RNA synthe-sized in thepresenceof100 ,uManisomycinwas isolated and separated according to molecular weight in a denaturingmethylmercury
hydrox-ide gel and immobilized on DBM-paper as de-scribedabove. Variousrecombinantplasmids
of the XbaI-Eregion (Fig.
1) orvarious
restriction enzyme DNAfragments from recombinant plas-mids were 32p labeled by nicktranslation
(41) and used asprobes
tolocate
theregions
of IE RNAtranscription.
The mRNAsize
classes were localized to the nearest restriction frag-ment or junctionof
twofragments. Figure
2illustrates
thesize
classesof
IE RNAdetected by the varioussections
of the XbaI-E region. The exposureperiod
of theautoradiogram
var-iedaccording
totherelative
concentration of theimmobilized
viral RNA.Transcription
from theBamHI-D region (data J.VIROL.on November 10, 2019 by guest
http://jvi.asm.org/
IMMEDIATE EARLY GENES OF HUMAN CMV CMV Towne
MapUnits
Components
XbaI
Bam Hi Sal I Sac I Pst I *Ava I *Sma I *Hinc 11
Map Units 0.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
I I I I 1.0
TRL UL IRL IIRS US TRS
M',0 A L K P a J C R(U)N E TOM2 H a I
liii II l~~~~~~iHii HiHI
ID C E A
D 4 C 4 B 4 A 4 E
B 4 A 4 D 4E c
pXS ~SUF G
A B EF4 D 4 c
44
...
*680 0.709 0.728'0.732 0.739 0.751
D=MH F=Ml
0.770
FIG. 1. Physical maps for the XbaI cleavage sites for human CMV(Towne)DNAandfortheBamHI, Sall, Sacl, andPstIcleavage sites for XbaI-EDNA.TheCMV genome(240kb) consists of197 kboflong unique (UL) sequencesand 42kb of shortunique(Us)sequences. Ateachend of thelonguniquesequences,therearerepeat sequencesof about11 kb thatareinverted relative toeach other. Likewise, ateach end of the shortunique sequences, there arerepeat sequences of about 2.0to2.5kb that areinvertedrelativetoeach other. Sucha structureresults infourequimolarpopulationsof the viralDNAdifferingin therelative orientations of thelong and short segments. The BamHI letters in parentheses representfragment designationsof the entire BamHI physical map (52) determined by La Femina and Hayward (submitted for publication). Regions ofXbaI-E subcloned into various recombinant plasmids aredesignated. Cleavage sites forAvaI, SmaI, andHinclI are designated only for definedregions within the XbaI-EDNA.Thedatafor theXbaI physicalmapof the entire
viral genomearefromLaFemina andHayward (27; submitted forpublication).
notshown), theBamHI-C region (Fig. 2,lane 1) and thePstI-C region (datanotshown) of XbaI-Ewasnotdetected.Threeseparateregionsof IE RNA transcription within the XbaI-E region
were detected. The approximate limits of each
region were defined as follows. A 1.95-kb IE
RNA sizeclass wasdetected withfragments of
viral DNA extending from the BamHI-E frag-ment into thePstI-B region, but not extending beyond theAvaI site (Fig. 2,lanes2, 3, and4). Thisregion encoded forasingle size class ofIE
RNA that was present in the infected cell at relatively low concentrations requiring 16 h for detection by autoradiography. This region was
designatedas IEcodingregion 3 (Fig. 2)and is located between0.709 to0.728 map units (Fig.
1).Finerrestrictionenzyme maps arenecessary
tonarrow the limitsforcoding region 3. Theregionfrom theAvaI siteinPstI-Btothe left end of the Sall-A region was either not transcribedorpartially transcribed (Fig. 2, lane 5).Itispossiblethatthisregioncontainsasmall amount of leader sequence for DNA coding
region 2described below.
The region from the left end of the Sall-A fragment to the leftend of the PstI-E fragment detectedfiveIERNAsrangingfrom 2.25to1.10
kb
(Fig.
2, lane 6). Twoof
theRNAs,
1.75 and 1.40kb,
wereslightly
more abundant than the rest. The RNAsfrom
thiscoding
region
werealso
presentin theinfected
cellatrelatively
lowconcentrations
requiring
16 h for detectionby
autoradiography.
The same RNAsize classes
weredetectedwith
32P-labeled
BamHI-BDNA,
exceptthatwith this
probe
a1.95-kb
size class of RNA washighly abundant
(Fig. 2, lane 7).
Aprobe
extending from
theleft
endof
PstI-E to theSmaI site in
thePstI-Eregion
alsodetected
a1.95-kb
RNA, butnotinhigh
abundance(Fig. 2,
lane 8).
Therefore,
theregion from
the left endof
the Sall-A
fragment
totheSmaI
site in the PstI-Eregion (0.732
to0.739 mapunits)
wasdesignat-ed
coding
region
2(Fig.
2).
The
predominant
IE RNA has asize class of 1.95kb andwaseasily detected afteronly
2 hofautoradiography (Fig.
2). The32P-labeled probe
from theSmaI
site in PstI-E totheright
endof
PstI-E(Fig.
2,lane9)aswellastheentire PstI-E DNA(Fig. 2,
lane10)
and PstI-F DNA(Fig.
2,
lane 11)detected thepredominant
1.95-kb RNA.Although
the same size class of RNA wasde-tected
withPstI-G,
the relative amount of hy-bridization waslower(Fig.
2, lane12).
Inaddi-tion,
thepredominant
IE RNA was noteasily
5 VOL. 46,1983
on November 10, 2019 by guest
http://jvi.asm.org/
[image:5.490.73.416.72.284.2]6 STINSKI ET AL.
FIG. 2. Autoradiograph ofNorthern blot hybridizations of IE polysome-associated poly(A) RNA. Virus-specificRNAwasdetectedby hybridization of32P-labeledviral DNApreparedasdescribed in thetext.Thesizes of the viral RNAs are indicated in kb. The autoradiograms were incubatedfor various periods of time as indicated. The following recombinant plasmids or DNA sections were used as
32P-labeled
probes in theindicated lanes: 1,BamHI-C; 2,SalI-C;3,left end ofSalI-Btoright end ofPstI-A;4,left end ofPstI-Btoright end of Sail-B; 5,AvaIsite inPstI-Btoright end ofSall-B; 6, left end ofSail-A
toright end ofPstI-B;7, BamHI-B;8,left end ofPstI-EtoSmaIsite; 9,SmaIsite in PstI-E toright end of PstI-E; 10,PstI-E;11,PstI-F;12,PstI-G;13, left end ofPstI-Dtoleft end ofSacI-E;14,SacI-E;15, right end ofSacI-EtorightendofPstI-D;16,PstI-D.Thevarious DNA-coding regions are designated as 1, 2, and3.detected by
a32P-labeled
probe from the right
endof
PstI-Gtotheright
endof SacI-D(Fig. 2,
lane 13). Because theseprobes
arerelatively
small,
haveintron
sequences,andarerelatively
rich
inadenine
andthymine (Stenberg
etal.,
unpublished data), they
may havehybridized
less
efficiently
with thepredominant
IE RNA. A32P-labeled
SacI-E DNAprobe (Fig. 2, lane 14)detected
thepredominant IE RNA withina 2-hincubation
period
forautoradiography.
Like-wise,
the3NP-labeled
probe representing the PstI-Dfragment
detected the predominant IE RNA(Fig.
2, lane 16). However, the regionextending from
the right end ofSacI-E
to theright
end ofPstI-D
was nottranscribed under IEconditions
(Fig.
2, lane 15).Therefore,
there-gion
extending
from the SmaI site in PstI-E to theright end of SacI-E (0.739to0.751 map units) wasdesignated
ascoding region
1(Fig. 2).
The mapunits of the various IEregions
arebasedondetermining
the size of the various DNA frag-mentswithin the XbaI-Eregion by usingawide range of DNA size standards.Electronmicroscopy ofR-loops withtheXbaI-E region. Electron
microscopy
ofR-loops
wasutilized
primarily
tovisualize
the threecoding
regions within XbaI-E and
secondarily
toquali-tatively estimate coding distances.
IEpolysome-associated, poly(A)
RNA was selectedby
hy-bridization
with XbaI-E DNA linked toDBM-cellulose.
After elution of theRNA,
the RNA wasethanolprecipitated
andsuspended in bufferon November 10, 2019 by guest
http://jvi.asm.org/
IMMEDIATE EARLY GENES OF HUMAN CMV
4~~~~~~~4
,.'.;'i,>.,"R .e ',, f "e s .' I ~~~~~~~~t~ _
t
v% 4FIG. 3. Electron micrograph of the XbaI-E DNA of CMV bearing R-loops formed with IE RNA. RNA complementarytoXbaI-EDNAwasselectedbyhybridization toXbaI-EDNAlinkedtoDBM-cellulose. The selected IE RNA washybridizedto1,ugofXbaI-digested,recombinantplasmidXbaI-E DNAasdescribed in the text. Size markers were double-stranded (ds) vector plasmid pACYC 184 and single-stranded (ss) phage M2 DNAs.Theinterpretative drawing illustrates the R-loops formed whicharedesignatedascodingregions 1, 2, and 3. The
hybrids
arerepresented
diagrammatically
by
the thickerline,
and thedisplaced single-stranded
DNA strandsarerepresented diagrammatically by the thinner line. The orientation forthemoleculewasfacilitatedby thelarge 3.0-kb R-loopwhich was positioned to the left.Magnification, x12,500.for
theformation of
R-loops with XbaI-E DNA asdescribed
above.Figure
3 shows threeR-loops within
the XbaI-E DNA thatare designat-edcoding regions
1,2, and 3. From the left-hand XbaI sitetocoding region
3, therewas approxi-mately 4.6 kb of DNA nottranscribed
under IEconditions.
TheR-loop
associated withcoding
region
3extendedfor
approximately
3.0kb. To theright of coding
region
3, therewas aregion of
approximately
2.8 kb that was not transcribed under IEconditions.
Coding
region
2extendedfor
approximately
1.7kb inadirection
opposite
thatof
coding
region
1.Thepoly(A)
tailsof
the mRNAsof
coding regions
1and 2wereestimated
tobeapproximately
230 basepairs
apart(Fig.
3).
Coding region
1extended
forapproximately
2.8kb,
but the exact measurement wasfrequently
complicated by loops
foundtobenearthe5'end 7 VOL.46,1983on November 10, 2019 by guest
http://jvi.asm.org/
[image:7.490.104.395.77.497.2]of the
mRNA. Finally, there wasagain
arela-tively
large region ofapproximately 4.7 kb to the right of region 1 that was not transcribed under IEconditions.
The measurements presentedabove
representapproximations subject
to thelimitations
of electronmicroscopy and
arebased
ondouble-stranded and
single-stranded
DNAsize standards described above. The
extentof
the
three encoding regions and the lack of
IEtranscription
at theleft
aswell
astheright ends
of the XbaI-E region
were ingood
agreement with the Northern blot analysis presentedin
Fig.
2.Direction of transcription by Southern blot analysis.
Electron
microscopy of R-loops
sug-gested that coding regions 1 and 2 weretran-scribed in
opposite directions. Southern
blothybridization
wasused to map the IE RNAsand
their
3'ends.
Virus-specific
IE RNA wasselect-ed
by hybridization
toXbaI-E
DNAlinked
toDBM-cellulose.
32P-labeled
cDNAstoeither
theentire
RNA sequence (total cDNA) or tothe 3'
...~~~~~~~~~~~~...:....
FIG. 4. Southern blot hybridization of
32P-labeled
total or 3' cDNA made to selected IE RNA. IE polysome-associated, poly(A) RNA was selected by hybridization to XbaI-E DNA linked to DBM-cellu-lose.32P-labeled
total or 3' cDNA probes to the selectedIE RNA weresynthesized withreverse tran-scriptaseasdescribed in thetext.32P-labeled recombi-nantplasmidXbaI-Ewasprepared by nick translation (41). Recombinant plasmid pCB42DNA(lane 1)was digested withthe restriction enzymesBamHI, PstI, andSall. Recombinant plasmid pMW34 (lane 2)was digested with the restriction enzymes BamHI and PstI. Samples of5 x105
cpmof32P-labeled
XbaI-E DNA (A), total cDNA (B), or 3' cDNA (C) were hybridized for48 h at42°C as describedinthe text. The sizesofthe viralDNAfragmentsareindicated in kilobases. The locations of the various restriction enzyme sites and their relationship to DNA coding regions1 and 2 areindicated.ends
of
the RNA sequence(3' cDNA)
were prepared as described above. Selected sub-clones of the XbaI-E region containing the BamHI-A (pMW34) or -B (pCB42)fragments
were digested with the restriction enzymes BamHI and PstI or BamHI, PstI, andSalI,
respectively (Fig. 1). After immobilization of DNAfragments of 2.0 kb or less by themethod of Southern (43), probes of 32P-labeled totalcDNA
or3' cDNA
aswell
as32P-labeled
XbaI-E DNA were used for hybridization as described above.The 32P-labeled recombinant plasmid XbaI-E
probe hybridized
to all DNAbands,
including
vectorplasmid DNA, with equalintensity (Fig. 4A). The 32P-labeled total cDNA probe hybrid-ized intenselyto a1.25-kb DNA (Fig. 4B, lane1) representing region 2 from the left end ofSall-A tothe left end of PstI-E (Fig. 1) and to a 0.75-kb DNA (Fig. 4B, lane 2) representing region 1 from the leftend of BamHI-Atothe right end of PstI-E (Fig. 1). In contrast, the relativeamount of hybridization with 32P-labeled total cDNA probe was less to a 0.70-kb DNA (Fig. 4B, lane 1) extending from the left end of PstI-E to the left end of BamHI-A (Fig. 1), which is theregion of DNA whereregion 1 encounters region 2(Fig. 2). The total cDNA probe also hybridized to a 0.60-kb DNA that is the PstI-F fragment of encoding region 1(Fig. 4B, lane 2).When the 32P-labeled 3' cDNA probe was used for
hybridization,
the relative amount ofhybridization
waslow to the 1.5-kb and the 0.75-kb DNAs representing coding regions 1 and 2, respectively (Fig. 4C, lanes 1 and 2). Thishy-bridization is presumably due
to asmall
amountof
32P-labeled
cDNAsynthesized from
RNAtemplate
that wasonly partially degraded.
In contrast, therelative
amountof
hybridization
washigh
to the0.70-kb DNA that is the regionof
DNA wherecoding region
1 encounterscoding
region
2(Fig. 4C, lane
1).Therefore,
the 3'ends
of the IE RNAs incoding regions
1 and 2 map near orwithin
the 0.70-kb DNA that extends fromthe left end of PstI-E DNA to the left end of BamHI-A DNA(Fig.
1). These results also confirm in part thelocation of
coding regions
1 and 2 identified by Northern blot hybridizationanalysis.
Thetranscription
ofcoding
DNA se-quence 1 proceedsfrom right
toleft,
whereastranscription of encoding
DNAsequence 2is
on the complementary strand and in theopposite
direction.
The 3' ends of these viral RNAs arelocated approximately
left and rightof
0.739 mapunits.
The exactlocation
of the 5' and 3' endsof the above genes iscurrently being
deter-mined
by DNA sequencing.The
physical
maps in coding region 3 werenot
defined
in sufficient detail to allowunambiguous
orientation
of this mRNA. Since this viral RNA VIROL.on November 10, 2019 by guest
http://jvi.asm.org/
[image:8.490.61.229.329.498.2]IMMEDIATE EARLY GENES OF HUMAN CMV 9
and its
protein product are synthesized in
rela-tively low amounts, the orientation of this
mRNA was not analyzed further.
In vitro translation products of IE RNAs. The
XbaI-N and -E regions of the viral genome are
located between 0.660 to 0.770 map units and
code approximately
88%
of the IE whole cell
RNA as
well
as for the
abundant size classes of
polysome-associated, poly(A) RNA (52). To
de-termine the translation products of these IE
RNAs, IE polysome-associated, poly(A) RNA
was
preparatively hybridized to XbaI-N or -E
DNA
linked to DBM-paper and subsequently
eluted. The
virus-specified RNAs were then
tested for translation in a rabbit reticulocyte
lysate as described above. RNA isolated when
using the
XbaI-N
DNA
did not translate in the
reticulocyte lysate. RNAs originating from this
region of the genome at various times after
infection are currently being investigated.
Prep-arations
of IE RNA selected by hybridization to
XbaI-E were translated. The virus-specified
RNA
eluted from
XbaI-E DNA was hybridized
to
XbaI-E DNA a second time, eluted, and
translated in vitro. Four
polypeptides
having
apparent
molecular weights of 75,000, 56,000,
39,000,
and 16,500 were identified
by
SDS-polyacrylamide
gel
electrophoresis
(Fig.
5,
lane
2).
Some polypeptides between 39,000 and
16,000 daltons
were present at relatively low
concentrations. The polypeptide of 75,000
dal-tons was the
predominant translation product.
To
determine the location on the genome of
the various
proteins
coded for by IE RNA, three
sections
of the XbaI-E DNA
representing
coding
regions 1, 2 plus 3, or 3 were used. Since the
Northern
blot
hybridization
(Fig. 2) and in vitro
transcription
with
Manley extracts (Thomsen et
al., unpublished
data)
indicated that the
predom-inant IE RNA for coding region 1 was initiated in
the
PstI-D
region (Fig. 1), this DNA as well as
the PstI-F, -G, and -D DNAs (Fig. 1) were
immobilized on DBM-paper for mRNA
hybrid-ization. In addition, the Sacl-A DNA and the
Sall-C DNA
(Fig. 1) were
immobilized
for
hy-bridization of viral RNAs originating from
cod-ing regions 2 and 3.
Since
the
Northern
blot
analysis
indicated that
coding region
1
washighly
transcribed under IE
conditions, whereas transcription from coding
regions 2 and 3 was relatively low, the amounts
of IE
poly(A)
RNAused for
hybridization were
20, 80, and 100 ,ug for coding
regions 1, 2 plus 3,
and
3,
respectively. The IE RNA was eluted and
translated in
vitro as described above.
The
IE RNAselected
by
coding region 1,
thePstI-D
DNA, or the PstI-F, -G, and -D DNA
(data
notshown)
wastranslated into a
predomi-nantpolypeptide of 75,000 daltons and a
poly-peptide
at lowerrelative
concentrations of
39,000
daltons (Fig. 5, lane 5). Several additional
polypeptides of lower molecular weight
werepresent that were not detected in the infected
cell;
consequently,
these
polypeptides may
notbe virus
specified. The nonspecific polypeptides
above 42,000
daltons
areendogenous
tothe
rabbit reticulocyte lysate, and some of these
areshown in
Fig. 5, lane 3. The
polypeptides below
39,000 daltons may be premature termination
products or endogenous products that
arenor-mally not detected unless the
reticulocyte
lysate
is
highly stimulated.
Alternatively,
these
prod-ucts
may
reflect
an
unusual property of the viral
mRNA that causes early
termination.
Polypep-tides of
similar apparent
molecular
weight
werenot
found
in
infected cells
(Fig.
6).
The IE RNA
selected by
hybridization
to
the
Sacl-A DNA was translated
topolypeptides
of
56,000, 42,000, 21,000, and 16,500 daltons
(Fig.
5,
lane 6) plus several
additional
polypeptides.
Infected
cell-specific
polypeptides of 56,000,
42,000,
21,000, and 16,500 daltons have been
detected in infected
cells
(Fig. 6)
under
IE
conditions,
but at
relatively
low levels
(6,
7, 45,
46,
53).
In
addition,
apolypeptide of 75,000
daltons
was
translated
(Fig.
5, lane
6). Our
current
measurementsof
the
predominant
IE
RNA
from coding
region 1 and our DNA
se-quence data indicate that the
majority of
tran-scripts
are
terminated before the Sacl-A site
MW11 xOo 1 2
75 _
56--
-39
3 4 5 6 1
16.5-.._ K7'
-56 .42 --39
--21
[image:9.490.253.444.403.572.2]-16.5
FIG. 5. Fluorograms of the in vitro translation products ofvirus-specified IE mRNA. Lanes: 1, in vitro translation with adenovirus type 2cytoplasmic RNA; 2, IE RNA selected twicebyhybridization to XbaI-EDNA; 3,noaddedRNA; 4, adenovirus type 2 cytoplasmicRNA; 5,IERNAselectedby hybridiza-tion to PstI-D DNA; 6, IE RNAselectedby hybridiza-tiontoSacI-ADNA; 7, IE RNA selectedby hybridiza-toSalI-CDNA.The apparent molecularweights of the virus-specified polypeptides coded for byIE RNA are designated.
VOL.46,1983
..4 A-W
...-,.-7' 7.','; .. , -1. 't :, S.,-S1.
on November 10, 2019 by guest
http://jvi.asm.org/
(Stenberg
et al.,unpublished derably
lower amount oftransl,
dalton
polypeptide by RNA fr eventhough fourfold
moreRI
hybridization
may be due to IA
1 2 3 4 5 &
4S*
lata). The
consid-
coding region 2,(ii)
occasional transcriptionation of
a 75,000- across theSacl-A
site from coding region 1, or omSacl-A
DNA(iii)
contamination by the abundant IE RNA NA was usedfor
from region 1. The IE RNA from region 1is20-(i) IE RNA from
to30-fold
moreabundant than the IE RNA fromregions 2 and 3 (Thomsen
etal.,
unpublished
data); consequently, it is difficult
to removeall
of this viral RNA from the DNA-DBM filter. In
c
Yv1'W.'
addition,
thepredominant IE
RNA translatesextremely well. We
areable
totranslate
aslittle
as10
to15
ngof this mRNA; consequently,
theslightest
amountof contamination
would be
de-tected in the rabbit
reticulocyte lysate. There is
currently
noevidence
to suggestthat there
are i...75similar
sequencesin
coding
regions
1and
2.2
Therefore,
wefavor the latter
interpretation for
the
presenceof this translation
product when
i:
-^
:using Sacl-A DNA for mRNA selection. The IE
RNA
selected by
hybridization
toDNA
repre-senting coding region 3, the SalI-C
DNA(Fig.
1), coded
for
a veryminor
polypeptide of
ap-proximately 68,000 daltons (Fig. 5, lane 7).
Immunoprecipitation of the predominant IE protein synthesized in vitro or in vivo. A mono-clonal antibody (E-3) against the 72,000-dalton IE
protein (19)
was used to immunoprecipitateIE
protein synthesized in vitro
orin
vivo.
The controls were normal mouse serumplus
lysate from in vitro translation or infected cells.Addi-2 4 w:
, --7115
72
-42
-39
[image:10.490.54.242.153.410.2]--21
FIG. 6. Fluorograms of IE polypeptides synthe-sized invitro or in vivo and immunoprecipitated by monoclonalantibody.(A) Lanes: 1,polypeptidesfrom in vitro translationwith adenovirus type 2cytoplasmic RNA; 2, in vitro-translated adenovirus polypeptides plus monoclonal antibody E-3; 3, in vitro-translated polypeptides of IE RNA selected byhybridizationto PstI-DDNA;4and 5, invitro-translated polypeptides of IE RNA selectedbyhybridizationtoPstI-D DNA plus normal mouse serum (lane 4) or monoclonal antibodyE-3(lane 5); 6, infectedcell-specific polypep-tides pulse-labeled with
[35S]methionine
in the pres-enceofactinomycinDaftertreatmentof theinfected cells for 12 h with cycloheximide; 7, infected cell-specific polypeptides pulse-labeled with [35S]methio-nine from1to4hpostinfectionin the absenceofdrug treatment;8, uninfected cellpolypeptides. (B)Lanes: 1,polypeptides from in vitro translation with adenovi-rustype 2cytoplasmic RNA; 2, uninfected cell poly-peptides; 3, uninfected cell polypeptides plus monoclonal antibody E-3; 4, infected cell-specific polypeptides pulse-labeledwith[35S]methioninein the presence of actimomycin D after treatment of the infected cells for 12 h withcycloheximide; 5 and 6, infected cell-specific polypeptides pulse-labeled and treatedasdescribed above plus normalmouse serum (lane 5) or monoclonal antibody E-3 (lane 6); 7, in vitro-translated polypeptides of IE RNA selectedby hybridization to PstI-D DNA plus monoclonal anti-body E-3. The apparent molecular weights of the infectedcell-specificIEpolypeptides and the immuno-precipitated polypeptidesaredesignated.J. VIROL.
....
on November 10, 2019 by guest
http://jvi.asm.org/
IMMEDIATE EARLY GENES OF HUMAN CMV 11
tional
controls
werespecific monoclonal
anti-body plus lysate from in vitro
translation of
adenovirus mRNA
oruninfected cells.
The IE mRNA selected by hybridization to
PstI-D DNA
representing coding region
1 wastranslated in vitro and analyzed by
SDS-poly-acrylamide
gel electrophoresis asdescribed
above. Figure
6A(lane
3)shows the
typical
polypeptide profile with the predominant IE
protein of 75,000 daltons, the minor polypeptide
of
39,000 daltons,
aswell
asthe
endogenous and
low-molecular-weight proteins described above
(Fig. 5, lane 5). Monoclonal antibody
E-3im-munoprecipitated the 75,000-dalton IE
polypep-tide,
but
notthe 39,000-dalton
polypeptide
(Fig.
6A, lane 5). Normal
mouse serumcaused
noimmunoprecipitation (Fig.
6A, lane 4).The
immunoprecipitated
IE75,000-dalton
polypeptide comigrates with
someof the IE
polypeptides found in the infected cell and
syn-thesized under IE conditions. However, the
proteins synthesized in vivo have
an apparentmolecular
weight
rangeof
75,000
to68,000
aspreviously
reported (46). The
majority
of
the IE
polypeptides
had
anapparentmolecular
weight
of
72,000
(Fig.
6A, lane 6).
Asimilar infected
cell-specific polypeptide
of 72,000 daltons
canbe detected in infected cells without
resorting
to acycloheximide block for mRNA accumulation
and
pulse-labeling in the
presenceof
actinomy-cin D
(Fig. 6A, lane 7).
Since
the
predominant IE protein
is 75,000
daltons
when
synthesized in vitro, but is found
in
vivo
as abroad banding protein of 75,000
to68,000 daltons,
monoclonal
antibody
E-3
wasused
toimmunoprecipitate
IE
protein
synthe-sized
in
vitro
orin
vivo,
and the
precipitates
wereanalyzed by SDS-polyacrylamide gel
elec-trophoresis.
Monoclonal
antibody
E-3 did
notprecipitate
anydetectable
proteins
from
unin-fected
cells
(Fig. 6B,
lane 3). In
contrast,the
monoclonal
antibody precipitated
the
predomi-nantIE
protein from
lysates
of cells
pulse-labeled under IE
conditions. The
apparentmo-lecular
weight of the protein ranged from 75,000
to68,000, with the majority of the protein
at72,000 (Fig.
6B, lane 6). The
majority of the IE
protein
synthesized in vivo migrated
slightly
faster
than the
predominant
IE
protein
of
75,000
daltons
synthesized
invitro
(Fig.
6B, comparelanes
6and
7). The
IE mRNA thatcoded
for
the IEprotein of 75,000 daltons originated from
coding
region
1.Therefore,
weproposethat the
IE
protein of primarily 72,000 daltons is also
coded
by region
1.The
causeof the
slight shift
inmigration
when
thepredominant
IEprotein
issynthesized in vivo
has notbeen
determined.Summary
of the IE RNAs andproteins coded within the XbaI-Eregion (0.685
to 0.770map
units).The
maplocations for
thevarious
coding
regions in XbaI-E, the direction of transcription,
the size classes
of IE RNAs, and the in vitro
translation products
aresummarized in Fig. 7.
IE geneexpression
of humanCMV is
dominatedby transcription of coding region
1.The
direc-tion of
transcription, where known, is indicated
by
arrowspointing toward the 3' end in the
prototype arrangementof the viral
genome.Re-gion
1codes
for
anabundant
mRNAof 1.95 kb
that is translated in vitro
to apolypeptide of
75,000 daltons. This
protein is presumably
modi-fied in
vivo
to aprotein of primarily 72,000
daltons.
Aninfected
cell-specific polypeptide
of
the
same apparentmolecular
weight is also
detectable
in the cell within
4h
postinfection
without manipulating
thecell
toaccumulate IE
mRNA
by
inhibiting de
novoprotein synthesis.
The
size of the minor polypeptides coded in
regions
1,2,
and
3 arealso shown. We
proposethat
coding region
1representsthe
predominant
region of viral
geneexpression
immediately
after
infection.
DISCUSSION
One
ormoreof the viral
proteins
coded
by the
IE mRNAs
of
herpesviruses
are necessaryfor
efficient
transcription
of the other viral
genes.This has been documented
for herpesviruses by
using wild-type virus in cells treated with
cyclo-heximide,
aninhibitor of protein synthesis (2, 9,
11-16,21, 23, 28-30,
39,48, 52, 53),
orwith
interferon (47). In addition,
mutantsof herpes
simplex virus that
are temperaturesensitive in
anIE
geneproduce
asimilar restricted
tran-scriptional
pattern
atthe
nonpermissive
tem-perature(36, 38, 54).
Five
major
IE mRNA
species
of
herpes
sim-plex virus
originate primarily, but
notexclusive-ly, from
the
inversely repeated
sequencesof
both the long and short
componentsof
the viral
genome(10, 11, 14, 22,
56). These mRNA
spe-cies accumulate in the
presenceof protein
syn-thesis inhibitors
atrelatively similar
concentra-tions.
In contrast,in the human CMV-infected
cell, IE mRNA originates
from
aregion (0.660
to0.770
mapunits)
in the
large
unique
componentof the viral
genome. Theregion (0.739
to0.751
mapunits)
designated
asDNA
coding region
1is
highly
transcribed relative
toregions
2(0.732
to 0.739 mapunits)
and 3 (0.709 to 0.728 mapunits).
Theregion of
0.660 to 0.680 mapunits
(XbaI-N) requires further investigation.
Itis
assumed that RNApolymerase
IIrecognizes
the promotersfor
IE RNAsynthesis.
We proposethat the
upstreamregulatory
sequenceof
DNAcoding region
1 competes moreefficiently for
RNApolymerase
IIand thatthis constitutes
thefirst
stepin
theregulation of
humanCMV
geneexpression.
In the
herpes simplex
virus-infectedcell,
at VOL.46,
1983on November 10, 2019 by guest
http://jvi.asm.org/
12
Map units: 0.680
DNA coding region:
Direction of
transcription:
0.109
0.7280.7320.(39
0.751 0.(703 , 2 1 1
@5 Ie
I
RNA size class(kb):
Translated Proteins(X103):
Immuno-precipitated protein(X103)
synthesized:
1.95
68
2.25,1.95,
1.75,
1.401.10
(75), 56 42, 21, 16.5
1.95
75.39
invitro: 75 invivo: 72
FIG. 7. Summary of the IE RNAs and proteins coded within theXbaI-E DNA region. The map units of coding regions 1, 2, and 3 depict the limits of the probes used to detect viral RNAs. The direction of transcription isindicatedfor coding regions 1 and 2. The thickness of the bar represents the relative abundance of the IE RNAs originatingfrom thevarious coding regions, estimated by the relative amount of hybridization and the incubation time oftheNorthernblotautoradiograms. The size classes of the viral RNAs are indicated inkilobases.The in vitro-translatedpolypeptides from IE RNA selected by the various coding regions are designated by apparent molecular weights(x103).Theabundant IE proteins synthesized in vitro or in vivo andimmunoprecipitated by monoclonal antibody E-3 are designated by apparent molecular weight(x103).
least
oneIE
protein has been
implicated
as aregulatory
protein necessary for the
efficient
transcription of
early
aswell
aslate viral RNA
(38,
55).
Since
the
72,000-dalton
protein of CMV
is the
predominant
IE
protein,
wehypothesize
that
this protein
plays the major role in
influenc-ing transcription
of the other viral
genesand,
consequently,
that the
protein presumably
has
aninfluence
in
determining
whether the
infec-tion is latent, persistent,
orproductive.
Immu-noprecipitation
analysis
of this
protein
syn-thesized in
vitro
orin
vivo
suggeststhat the
predominant IE protein is
synthesized
as a75,000-dalton
polypeptide
thatis
modified in
vivo in
a waythat
increases migration in
adenaturing
gel.
Gibson (18) has
suggested that
the
defused
natureof the predominant
IEpro-tein is
suggestive of structural
orconformational
heterogeneity. This could be
due tointer-
orintramolecular interactions (e.g., disulfide
bond-ing)
orrapid post-translational modification
(e.g.,
phosphorylation).
Thepredominant
IEprotein of CMV (Towne) is
phosphorylated (18).
Thepredominant
IEgenedoes
notappear tobe
located in
anactive genetic element
resulting in
different locations
ororientations within the
viralchromosome.
Inaddition,
themajority of
theviral
RNAfrom the predominant
IE genefollows
arepeatable
splicing
pattern(Stenberg
etal.,
unpublished
data). Since the translation of
thepredominant
IE mRNA invitro renders
aprotein that
migrates
morehomogeneously,
we proposethat the
broad
migrating
protein
seenin
vivo is due
topost-translational
modifications.
IE
antigens of CMV accumulate in the
nucleusof infected
cells(33,
49), but if the cells
aretreated with
cycloheximide
the
antigens
also
accumulate in the
cytoplasm (Landini and
Stinski, unpublished
data). In the
nucleus,
the
predominant
IE
protein
is
associated
preferen-tially
with
chromatin
(S.
Michelson,
personal
communication).
At
veryearly
stagesof
infec-tion, CMV induces
aprotein that is responsible
for
the stimulation of
chromatin
template
activi-ty asmeasured
by the
incorporation of [3H]UMP
in the
presence of
Escherichia coli RNA
poly-merase(24).
Therefore,
weproposed that the
predominant IE
protein of CMV is
aregulatory
protein that influences
transcription.
This
pro-tein
may serveobligatory
functions that
arerequired throughout the
replication cycle of the
virus.
The mRNAs and
proteins from
coding
regions
2 and3
weredetected under the conditions
defined
asIE.However, it is
possible
that these
areearly
genesthat
function after the
synthesis
of the
predominant IE
protein.
There
is
aswitch
from restricted
toextensive
transcription
after
synthesis of the predominant IE protein. Theregion of
the viral genome mostaffected is the
large
repeat sequenceand
adjacent
sequences(12, 52). Since viral
RNAappears on polyribo-J. VIROL.on November 10, 2019 by guest
http://jvi.asm.org/
[image:12.490.49.437.58.274.2]IMMEDIATE EARLY GENES OF HUMAN CMV 13
somes as
functional
mRNA
from
the above
regions at early
times
or
in the presence of
phosphonoacetic acid,
aninhibitor of
viral DNA
synthesis, we have
proposed that this
region
codes for
early viral
proteins
(52, 53). The data
suggest that the
predominant
IE
protein of
CMV
regulates transcription
from
the
above
region
as
well
as
other
regions
of
the
viral genome. The
predominant
IE
protein
may
enhance promoter
recognition by
interacting with the viral
chromo-some or RNA
polymerase
II. Even
though
the
predominant
IE
protein
may serve to
enhance
early
or
late gene
transcription, additional
regu-latory events are
presumably
necessary
for
transport
of
the
viral RNA
tothe
cytoplasm.
Most
of
the
viral
RNA
originating
from the
large
and
small
unique
sequences
is retained in the
nucleus at
early times
and not
transported
tothe
cytoplasm
until after viral DNA
replication (52).
The
above regulatory
events
presumably play
animportant role
in
the
protracted replicative
cycle
of human CMV and in the
species
restrictions
onreplication.
ACKNOWLEDGMENTS
Thisinvestigationwassupported byPublic Health Service grant2ROlAI13526from the National Institute ofAllergyand Infectious Diseases and by grant 1-697 from the National FoundationMarchof Dimes. M.F.S. istherecipient of Public Health Service career development award lK04A1100373 fromtheNational Institute ofAllergy and Infectious Diseases.
LfTERATURECITED
1. Anderson, K.P., R. H. Costa, L. E. Holland, and E. K. Wagner. 1979.Isolation and localization of herpes simplex type 1mRNA. J.Virol.30:805-820.
2. Anderson,K.P.,R. H.Costa, L. E.Holland,andE. K. Wagner. 1980. Characterization of herpessimplex virus type 1 RNApresentin the absence of de novoprotein synthesis.J.Virol. 34:9-27.
3. Bachl, B.,and W. Arber.1977.Physical mapping of BglII, BamHI,EcoRI, HindIII,andPstI restrictionfragmentsof bacteriophagePI DNA. Mol.Gen. Genet.153:311-324. 4.Bailey,J.M.,andN.DavIdson.1976.Methylmercuryas a
reversibledenaturingagentforagarosegel electrophore-sis. Anal.Biochem.70:75-85.
5.Bhbop, D. H.L., J. R. Claybrook, and S. Splegelman. 1967.Electrophoretic separation ofviral nucleicacidon polyacrylamide gels.J. Mol.Biol.26:373-387.
6.Blanton, R. A., and M. J. Tevethia. 1981. Immunoprecipi-tation of virus-specific immediate-early and early poly-peptides fromcellslyticallyinfectedwith human cytomeg-alovirus strainAD169. Virology112:262-273.
7.Cameron, J. M.,andC. M. Preston. 1981.Comparisonof the immediateearly polypeptides ofhuman cytomegalovi-rusisolates.J.Gen. Virol. 54:421-424.
8. Chamberlain,J. P. 1979.Fluorographicdetection of radio-activity in polyacrylamide gels with the water-soluble fluor,sodiumsalicylate. Anal. Biochem. 98:132-135. 9. Chua,C.C.,T.H.Carter,andS. St.Jeor. 1981.
Tran-scription of the human cytomegalovirus genome in pro-ductivelyinfected cells. J.Gen. Virol. 56:1-11. 10. Clements,J. B., J. McLauchlan, and D. J. McGeoch. 1979.
Orientation ofherpes simplex virus type 1 immediate earlymRNA's. Nucleic AcidsRes.7:77-91.
11. Clements, J. B., R. J. Watson, and N. M. Wflkie. 1977. Temporal regulation of herpes simplex virus type 1 tran-scription:location oftranscriptsontheviral genome.Cell
12:483-496.
12. DeMarcK4,J. M. 1981. Humancytomegalovirus DNA: restrictionenzymecleavage maps and map locations for immediate-early, early,and late RNAs.Virology 114:23-38.
13. DeMarchl, J. M., C. A. Schmidt, and A. S. Kaplan. 1980. Patterns of transcription of human cytomegalovirus in permissivelyinfected cells. J. Virol. 35:277-286. 14. Easton, A. J., and J. B.Clements. 1980. Temporal
regula-tion of herpes simplex virus type 2 transcripregula-tion and characterization ofvirusimmediateearlymRNA's. Nu-cleic Acid Res. 8:2627-2645.
15. Feldman, L., F. Rhon, J. H. Jean, T. Ben-Porat, and A. S. Kaplan.1979.Transcriptionof thegenome of pseudora-bies virus(aherpesvirus) is stringently controlled. Virolo-gy 97:316-327.
16. Feldnan, L. T., J. M. DeMarchl, T. Ben-Porat, and A. S. Kaplan. 1982.Control of abundance of immediate-early mRNAinherpesvirus (pseudorabies)-infected cells. Vi-rology 116:250-263.
17. Furukawa,T., A.Floret,andS. Plotkin. 1973.Growth characteristics ofcytomegalovirus in human fibroblast with demonstration of protein synthesis early in viral replication. J. Virol. 11:991-997.
18. Gibson, W. 1981.Immediate-early protein ofhuman cyto-megalovirus strains AD169, Davis, andTownediffer in electrophoretic mobility. Virology112:350-354. 19. Goldstein,L.C., J.McDougall,R.Hacluan,J. D.
Mey-ers, E. D.Thomas,andR. C.Nowinskd.1982. Monoclonal antibodiestocytomegalovirus: rapid identificationof clin-icalisolates andpreliminary use in diagnosis ofCMV pneumonia.Infect. Immun. 38:273-281.
20. Ho, M. 1982.Cytomegalovirus biology and infection, p. 131-212.InW.B. Greenoughand T.C. Merigan(ed.), Currenttopicsin infectious disease. PlenumPublishing Corp.,NewYork.
21. Jean, J. H., T. Ben-Porat, and A. S. Kaplan. 1974.Early functionsof the genome ofherpesvirus.III.Inhibition of thetranscription of the viral genome in cells treated with cycloheximide earlyduringthe infective process. Virolo-gy59:516-523.
22. Jones, P. C., G. S. Hayward, and B. Rolzman. 1977. Anatomy of herpes simplex virusDNA. VII. RNA is homologousto noncontiguous sitesin both the L andS componentsofviral DNA. J.Virol.21:268-276. 23. Jones, P. C., and B.Roizman.1979.Regulation of
herpes-virus macromolecular synthesis. VIII. The transcription programconsists ofthreephases during whichboth extent oftranscriptionand accumulation ofRNAin the cyto-plasmareregulated.J.Virol.31:299-314.
24. Kamata, T., S. Tanaka, and Y. Watanabe. 1978. Human cytomegalovirus induced chromatin factors responsible forchangesintemplateactivityandstructureofinfected cell chromatin.Virology90:197-208.
25. Kessler, S.W.1975.Rapidisolation ofantigensfrom cells with a staphylococcal protein A-antibody adsorbent: pa-rametersof the interaction ofantibody-antigencomplexes withproteinA. J. Immunol.115:1617-1624.
26. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature(London)227:680-685.
27. LaFemina, R. L., and G. S. Hayward. 1980. Structural organizationofthe DNAmolecules fromhuman cytomeg-alovirus,p. 39-55.InB. N.Fieldo and R. Jaenish(ed.), Animal virusgenetics. AcademicPress, Inc., New York. 28. Leung,W.C.,K.Dimock,J.R.Smiley,andS.Bacchetti. 1980. Herpessimplex virus thymidine kinasetranscripts
areabsent from both nucleus andcytoplasm during infec-tion in thepresenceofcycloheximide. J. Virol. 36:361-365.
29. Mackem, S.,andB.Roizman. 1980.Regulation of herpes-virus macromolecular synthesis: transcription-initiation sites and domains of aL genes. Proc. Natl. Acad. Sci. U.S.A. 77:7122-7126.
30. Macken,S.,and B.Rdzman. 1981.Regulationof herpes-VOL.46,1983