Analysis of the herpes simplex virus type 1 OriS sequence: mapping of functional domains.

0022-538X/91/084359-11$02.00/0

CopyrightC)1991, AmericanSociety forMicrobiology

Analysis of

the Herpes Simplex Virus Type 1

Oris

Sequence:

Mapping

of

Functional

Domains

DAVID W. MARTIN, SWATI PALIT DEB, JENNIFER S. KLAUER, AND SUMITRADEB* Department ofMicrobiology, University of Texas Health Science Center,

7703 Floyd CurlDrive, San Antonio, Texas 78284-7758 Received25January 1991/Accepted7 May 1991

The herpes simplex virus type 1 (HSV-1)

Oris

region resides within a 90-bp sequence that contains two bindingsitesfortheorigin-binding protein (OBP),designatedsites IandII.AthirdpresumptiveOBP-binding site(III)within

Oris

hasstrongsequence similarity to sites I and II, but nosequence-specificOBPbindinghas yetbeendemonstratedatthissite. Wehavegenerated mutations in sitesI, II,and III anddetermined their replication efficienciesin atransientin vivo assay in thepresence ofahelper virus. Mutationsinany one ofthe sites reduced DNA replication significantly. To study the role of

Oris

sequence elements in site I and the presumptive site III in DNA replication, wehave also generated a seriesof mutations that spanfrom site I acrossthe presumptive bindingsite Ill. These mutants were tested for their ability toreplicate and for the ability to bind OBP by using gel shift analyses. The results indicate thatmutations across site I drastically reduce DNA replication. Triple-base-pair substitution mutations that fall within the crucial OBP-binding domain, 5'-YGYTCGCACT-3' (whereYrepresents C or T), show a reduced level of OBPbindingandDNA replication. Substitution mutations in site I that are outside this crucial binding sequence show a more detrimentaleffectonDNAreplicationthan on OBPbinding. This suggests that these sequences are required for initiation ofDNA replication but are not critical for OBP binding. Mutations across the presumptive OBP-bindingsite III also resulted in a loss in efficiency of DNAreplication. Thesemutations influencedOBP binding to

Oris

in gel shift assays, even though the mutated sequences are not contained within known OBP-bindingsites.Replacement ofthewild-type site III with a perfectOBP-bindingsite Iresults inadrastic reduction ofDNAreplication.Thus, our DNAreplicationassays and in vitro DNA-binding studies suggest that thebinding oftheoriginsequencebyOBP is not the onlydeterminingfactor forinitiation of DNAreplication invivo.

Thegeneral modelof initiationof DNAreplication

emerg-ing from studies on prokaryotic and eukaryotic systems indicates that most sequence-specific initiations start with therecognitionof and bindingtotheorigin ofreplicationby an initiatorprotein (2). Thisbinding signals the subsequent steps whichinvolvelocalizationof other protein factors such ashelicases and DNApolymerases, etc. The recognitionof the origin sequence by the initiator and the subsequent protein-protein interactions are of very high precision and result in aspecialized nucleoprotein structure. Herpes

sim-plex virus type 1 (HSV-1) is an attractive model system in

which to study the initiation of viral DNA replication.

Presumptive origins of DNA replication have been mapped on the viral genome, and the virus encodes many of the

proteins required for initiationofDNA replication. Thus, a study ofthe mechanism of initiation can be undertaken by

usinggenetic andbiochemical approaches.

HSV-1 has alinear,double-stranded DNAgenome of 152

kbp(21). Definitivemapping of viral DNA replication origins was accomplished by studying the replication ofdefective interfering particles (12-14, 16, 17) and cloned virus DNA segments in the presence of helper virus (23, 25, 27, 29). ThreepresumptiveoriginsareontheHSVgenome: two

Oris

are present as one copy in each c-repeat of the viral chromosome (27), and a third origin, designated OriL, is associated with theunique long (UL) region (29).

Sevenvirally encoded genesarenecessaryfor viral DNA

synthesis (22, 31). Elias et al. (11) identified a factor (the

* Correspondingauthor.

origin-binding protein or OBP) from infected cell extracts that could sequence specifically bind to

Oris.

OBP was

subsequentlyshownbyOlivoetal.(24)tobeencodedbythe UL9 gene and is one of the seven virally encoded genes essential for DNA replication. We (3) and later others(15,

28) have shown thatoriginbindingofOBPcanbecorrelated

to origin function. Because of its specific origin-binding

function, OBP may represent the initiator protein for viral DNAreplication.

The

Oris

sequencecontainsa45-bpimperfectpalindrome

with a central A-T-rich stretch. Previous analyses have

demonstratedthe existenceoftwo OBP-bindingsites in this

region(10, 24).Byusing site-directed mutagenesis(3,9) and

methylationinterference (18), it has beendemonstrated that the OBP recognition site is included within the sequence

5'-YGYTCGCACT-3' (whereY representsCorT). The

Oris

region alsocontains asequence which hasstrong sequence

similarityto OBP-binding sitesI and II, designated siteIII.

No

sequence-specific

OBP binding, however, has yet been

demonstrated to this site (9, 28). Thus, the role of this sequence in any

Oris-OBP

interaction is not established.

Analysis oforiginsequences inherpes simplex virus type 2 (HSV-2) has shown that deletion of part of a sequence

correspondingtosite III in HSV-1resulted inadramaticloss in DNA replication (20). Recent analysis in HSV-1, how-ever, has shown that deletion of site III affects

replication

efficiency onlymoderately (28).

We have mutagenized sites

I,

II,

and III.

Replication

efficiencies of these mutated

origins

demonstrate that for

optimal

replication

all the three sites are

required.

We have 4359

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4360 MARTIN ET AL.

A. |AGCTTOOCCGCCGOOTAAAAGAAGTGAGAACGCO E CGTTCGCACTTCGTCCCAATATATATATTAT |TAGOCGAAGTGCGAOCACTOGCGCCGGCCCCGGGC

IACCGGCGGCCCATTTTCTTCACTCTTGCGCTTCGCA AGCOTGAAGCAGGGTTATATATATATAATAATCCCO ICTTCACGCTCGTGACCGCGGCCGOOOCCCGGTAC

PAUNDROME

B.

III

I

II

5'-CGGGT AAGAAGTGAGAACGC X GCGTTCGCACTTCGTC AATATATATATATTATT

AGGCGAAGTGCGAGCACGrGCGCCGGCCCCGG-3'wildt

3'-GCCCA TTCTTCACTCTTGCGqCGCAAGCGTGAAGCAGGTTATATATATATAATAA CCGCTTCACGCTCGTG^CCGCGGCCGGGGCC-5 YP

TTT bs-7

TAC bs-8

TCC bs-9

CCT bs-1O

ATA bs-11

GG bs-12

ATA bs-13

GGT bs-14

AGA bs-15

GAG bs-17

GCCCATTTTCTT ACTCTTGCGCTTCGCAAGCGTGAAGCAGGGTTATATATATATAATAATCCCGCTTCACGCTCGTGACCGCGGCCGGGGCC 16 del

A

GCAACTTTTCTTCACTCTTGCGCTTCGCAAGCGTG AGCAGGGTTATATATATATAATAATCCCGCTTCACGCTCGTGACCGCGGCCGGGGCC bs-18del

AAA A

FIG. 1. Construction ofOris and its mutants. (A) Wild-type Oris constructed from 6 cassettes of overlapping oligonucleotides. The synthetic origin is cloned in the pOR vehicle (8). (B) Mutations were introduced by using cassette-directed mutagenesis. Overlapping oligonucleotidescontaining the desired sequences werehybridized, and theresultingcassettewasligated into a pOR vector. Forsubstitution mutants, Aresidues were exchanged for C and G for T. A single strand of the mutations is shown thatcorrespondstothe bottom strand(3'

to5') ofthewild-typeOris sequence. For 16del and bs-18del, thepositions of mutationsareindicatedbyarrowheads.

also performed base substitution mutagenesis of the region spanning site I through site III. These mutants have been assayed for their ability to support DNAreplication and to bind OBP. Our results indicate that the crucial OBP-binding domain at site I is required for both DNA replication and OBP binding; flanking nucleotides are essential for the initiation of DNA replication but are not critical for OBP

binding. Sequences in site III are essential for efficient replication and can modulate OBP bindingat

Oris.

MATERIALS AND METHODS

Plasmidcloneconstruction and mutagenesis. Construction ofwild-type and mutant

Oris-containing

plasmids has been describedpreviously (6). Origin sequences have been cloned between the HindIII and NcoI sites of a pOR vehicle (8) which consists of pML2 nucleotides 651 to 4361 with a

HindlIl linker added to the 651 site and a polylinker (NcoI,

Sall, BamHI, andXmaI) added to theEcoRI site at nucle-otide 4363. Recombinant origins were constructed by using cassettes ofoverlapping oligonucleotides as described

pre-viously (5-7) (Fig. 1). Cassettes ofoverlapping

oligonucleo-tideswerehybridizedand thenligatedinto thepORvehicle. This mixture was usedto transform Escherichia coli DH5.

Ampicillin-resistant colonies were picked and screened for the mutations by Sanger dideoxy sequencing of both strands. Deletion (del) mutant 16 del and base substitution (bs) mutant bs-18 del were fortuitous isolates encountered

whilescreening for other mutants. For substitution mutants, A residues were exchanged for C and G residues were exchanged for T. The desired plasmids were purified by

cesium chloride-ethidium bromidedensitygradient centrifu-gation.

DNAreplication assay.Transient in vivoreplicationassays were performed to assess the function of mutant origins. Briefly, subconfluent Vero cells in 100-mm2 plastic dishes weretransfected with1,ugofsupercoiled testplasmid by the calciumphosphate precipitation techniqueasdescribed ear-lier (6, 7). At 4 h posttransfection, cells were shocked for2 minwith15% glyceroland washed with Hanksbalanced salt

solution, and the medium was replaced. Infection was started 6 h posttransfection with 5 to 10 PFU per cell of

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4361

PALUNDROME

III I 11

CGCGTTAGAACGMTAGAAC IWCG¶ICGCA G-CCAATATATATATAICATT =CGAG5WGA^CA >cGcccG

GCCCAIX7lCA AAG AGlTATATATATATAATAACCCGC ACGCGTGAI:;CGCGGCCGC wildtype

CGGGTAATAGAAGTGAGAACGCG

GCCCAITA¶ICACTC'IGCGC

TCGCACGCxGTCCCAATATATATATATTATTAGGGCGCrGKAGC GAGACrGCGGCCGGCCCCGC;

AGCGUGAAGCAGGATATATATATAATAATCCCGCTTCACGCTCGTI ACCGGCCGGGGCC

CGGGrAAAAGAAGTGAGAACGCGAAGCIGCACrGTCCCAATATATATAT TrrA GCCCAimI-iACTTACITCGCCGCAAGCGTCAAGCAGGGTTATATATATATAATAAT

CmGrGAAAAGA

AGlfi>tCGCGAAGCGTTCGCACTTCGTCCCcAATATATATATATATTAsGCGCGAAGWCrG

AWACTGGCGCCGGCCCCGG GCCCAn:C q)CGCTCCAAGCGTGAACEAGGGrr TTTATATATATATATiU,"-CCGC-1- U.A,CA!C'{ 1rC;.M CCC(C; CECC

GCGTICGCACTTCGTCCCAATATATATATATTATTA

CGCAAGCGIGAAGCAGGGTTATATATATATAATAAT

A.

*0

1-1

LOm

1-1

ir-- I

-CL) cl) CL) -0 .0 'D

>- I

-=0

4 Progeny l w

9.

B.

(2) "'

.0

1.0_

0.j-a

C)

.C

32

0

CY)

z0

HSV-1 strain KOS. Incubations were at 37°C. Cells were

harvested 16 hpostinfection. The resulting cell pellet was

lysed in 10 mM Tris (pH 7.0)-100 mM EDTA-0.5% sodium dodecyl sulfate(SDS)-proteinase K (100 ,ug/ml) at 50°C for 12 h. This was followed bytwo phenol-chloroform extrac-tions and onechloroform extraction. Theresultingaqueous

fractionwasethanolprecipitated and digested with RNase A

(10 ,ug/ml) for 1 h at 37°C, followed by a second ethanol

precipitation. Samples of the resulting DNA were digested

with BamHI and DpnI. BamHI linearizes the plasmid, while DpnIcleaves5'-GATC-3' sequences onlywhen theadenine

ismethylated. Since plasmids aregrownindam-positiveE. coli DH5, the adenine is methylated, and the resulting

sequences are cleaved by DpnI. Replicated DNA is not methylatedatthesepositionsandisnotcleavedby DpnI.As

aresult, replicated DNA remainsasunit-length monomers,

while input DNA is digested into many small fragments. DigestedDNAswereelectrophoresedina0.8%agarosegel.

Gels wereblottedonto GeneScreen Plus (DuPont) by alka-line transfer and then hybridized with nick-translated pBR322DNA.The blotwaswashed, dried,andexposedfor autoradiography. Autoradiograms exposed with no

intensi-FIG. 2. Replication of site I, site II, and site III mutants. Wild-type and mutant plasmids were transfected into Vero cells, followed by infection with HSV-1 strain KOS. The DNA was

isolated and digested with DpnI and BamHI, runonanagarosegel, Southern blotted, andprobed with nick-translated pBR322. Muta-tions tested are shown above. (A) Autoradiogram of replication assay. The progeny(replicated DNA) and input DNA are shown. Notetheloss in replication in del I (site I mutant), pORS-1 (deletion of siteII), bs-15(triple-base-pair substitution in site III), and del 31 (deletion of sites II and III).(B) The histogram shows the relative

replication efficiencies of themutantsversus wild-typeOris. Auto-radiograms of the replicationassay werescannedby laser

densitom-etry,andthesignalwasnormalizedtowild-type levelsonthe basis

ofinput (the largestDpnI-generated fragment).

fiers wereused for laserdensitometric scanningtocompare

therelativeefficiencyofreplication of the variousconstructs after normalization of theinput relative towild-type levels. Asa measure of the input DNA,availableas atemplate for

DNA replication, wehave used the largest DNAfragment arising by DpnI digestion. This serves as a control for our

transfection.

Generation of invitro-synthesizedOBPderivative.Deb and Deb (3a) recently demonstrated that the DNA-binding domain ofOBP is contained within a 269-amino-acid

seg-ment near the C terminus. A C-terminal 318-amino-acid

fragment of OBP covering the DNA-binding domain was,

therefore, used for DNA-binding studies. This fragment is designated del 1-534 and retains the sequence-specific DNA-binding activity of wild-type OBP (3a). To generate the protein, a BamHI (21655)-EcoRV (21463) fragment of the OBP gene was used. A NcoI linker was added to construct an initiator codon at the BamHI site. This frag-ment wascloned into a pGEM3 vector downstream of the SP6promoter. Ethidium bromide-cesiumchloride gradient-purified plasmid DNA was linearized by EcoRI, phenol-chloroformextracted, andethanolprecipitated. Transcripts

weresynthesized bySP6 polymerase followingtheprotocol

supplied by Promega. The DNAtemplate wasremoved by treatmentwith RNase-free RQ1 DNase (Promega), phenol-chloroform extracted, and ethanol precipitated. The

syn-thetic RNA was then translated in vitro by using rabbit reticulocyte lysates (Promega) in the presence of

[35S]me-thioninetolabel thenascentprotein accordingto the

manu-facturer'sprotocol.

DNA-bindingassays.(i) Filter-bindingassay. Filter-binding competition experiments were performed to determine the specificity of the binding of in vitro products with origin

sequences. The in vitro translation mixture was incubated with 32P-labeled

Oris

probe on ice for 30 minin a binding

del-1

pOR-SI

bs-15

del-31

'i,

I

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AL.

PAUNDROME

III

I

II

5-CGGGTAAAGAAGTGAGAACGCG GCGTTCGCACTTCGTCC AATATATATATATTATT

GGCGAAGTGCGAGCACT8GCGCCGGCCCCGGw3

t 3GCCCA TTCTTCACTCTTGC CGCAAGCGTGAA TTATATATATATAATAAQCCCGCTTCACGCTCGTG

CCGCGGCCGGGGCC-CCT ATA GG ATA

TTT TAC TCC

bs-7 bs-8

bs-9 bs-10

bs-1l bs-12 bs-13

1.0

0)

.5

w

0.5

0

0)

cc

CN '0" )M N

1L

I I IrO C

co) C)l)m -0

n0 .0 .0

FIG. 3. Replication of site I mutants. Wild-type and mutant

plasmids were transfected into Vero cells, followed by infection with HSV-1 strainKOS. The DNA wasisolated and digested with DpnI and BamHl, run on an agarose gel, Southern blotted, and probed with nick-translated pBR322. Mutations tested are shown above. Histogram shows relative replication efficiencies of site I

mutants versus wild-type Oris. Autoradiograms of the replication assay were scanned by laser densitometry, and the signal was

normalizedtowild-type levelsonthe basis ofinput.

III

I

CGGGTAAAAGTGAGACGCCfAGCGTICGCACTrCGTIC4AATATATATATATTATTA

GCCCAI mACr T TATAATAAT

CCT ATA

rrI TAC

TCC

0) D.,

C) C) C)

I I I

0c

0.0.0.

I >%

03

FIG. 4. ReplicationofpOR-Slmutants.Wild-type,pOR-Sl,and

pOR-Slmutantsweretestedaspreviously described (seeMaterials andMethods andthelegendtoFig. 2). Mutations testedareshown

above. The histogramshows thelevel of DNA replication ofthese

mutants relativetothewild type. pOR-Sl

pOR-Sl pOR-Sl pOR-Sl pOR-Sl

pOR-Sl bs-7 bs-8 bs-9 bs-10 bs-11

v

O

CO I Q

c 0

z

LU

c 0

1;1

.2

OL

cc11 T10 Ici

cc11

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Oris

A.

Wild-type OBP

Del 1-534

(A

0

CL 0:

cr c C)

tO

ar a

CL et

I O. Oa

CI.

E

I I I

Sf.Lew

1 13 1 2 12 364534 686 804 851

1 534

4,

FIG. 5. (A) Lines showing schematically the wild-type and del 1-534genes. The solid bar represents amino acid sequences remain-ing; the hatched bar represents amino acid sequences deleted. Some ofthe amino acid positions are indicated by numbers. (B) Filter-binding competition experiments showing Oris-specificbinding by thein vitro-synthesized del 1-534. The in vitro translation mixture wasincubatedwith32P-labeledOrisprobe as described inMaterials andMethods. Afterincubation, the mixtures were passed through nitrocellulose filter papers and washed. The bound DNA was countedby Cerenkov countingandthen eluted fromthefilter and loadedonto a5% nativepolyacrylamide gel. DNA was visualized by autoradiography. Each lane contained 3 ,u1 of in vitro-translated product,labeledOris,and theindicated amount of competitor DNA. Note that the inhibition by self-competitor (2- to 10-fold) was significantlygreater than thatbynonself-competitor(0- to2.5-fold).

PAUNDROME

III

I

II

5-CGGGT AAAGAAGTGAGAACGC GCGGTTCGCACTTCGTCt AATATATATATATTATTA

GGCGAGTGCGAGCACGGCGCCGGCCCCGG-3,

wildtype

3'-GCCCA TTCTTCACTCTTGCG CGCAAGCGTGAAGCAG TTATATATATATAATAA CCGCTTCACGCTCGTGA CGCGGCCGGGGCC-5

CCT ATA GG ATA

TTT TAC TCC

bs-7 bs-8 bs-9

bs-10 bs-11 bs-12

bs-12

aL a

0

DO CO CO DCO U)D Ca = .0 .0 .0 .0 .0 .0 .0 3' .

I, + + t + + + + -I Protein

e`e 4Complex I

4Complex 11

4 Free DNA

B. *Complex I

7Complex I I

FIG. 6. Gel shift analysis of site I mutants. del 1-534 was

incubated withwild-typeormutantOrisprobefor 30minat4°Cas

described in Materials and Methods. Theresulting complexeswere

resolvedon anative 5%polyacrylamide gelasdescribedin Materi-alsand Methods. Mutants testedare shown above.(A) Autoradio-gram ofgelshiftanalysis. NotecomplexI (OBPbindingtosites I

andII)andcomplexII(OBP bindingtosites IorII)formation in the wild-type lane. Note alterations in theintensityof thesecomplexes with mutantorigins. (B)HistogramshowinglevelsofcomplexIand II formation ofmutantorigins relativeto wild-typeOris. Autorad-iogramsofgelshiftanalysiswerescannedbyusinglaser densitom-etry.Resultingvalueswere normalizedtowild-typelevels. B.

bs

*a

_u _ec

-c

CD

m

CD

CC

c)C ' 0o )cn 0D .. ) II

a) CO J 9 COn

DO QO

_0 .0 .0 J

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4364 MARTIN ET AL.

III I

CG(k;tP?r AAGTlGAGAACGCcGCGFA ICGCAC¶[TCGr2 'AATATATATATATTATTA

GCCCA ItI'II\CruGlC?r rCaAAGCGCTAAACA TTATATATATATAATAAT

CCT ATA

TAC TCC

I

co co

-0 0

I I

Q Q

a

- L

CY)

I .0

CO

0._+L

00 N c) X) in

'r- 1rl*

CO CO

I

0 0

.L

+ + + I

0 0

0.0.L

1 4

Protein

AComplex

I

4Free

DNA

B. r_

-2

m

m

-Z1

v- 0

'r-I V-I

0)

nl

CO

I~ I

co cc

0 0

CL

Ca) OCo

I,,-l I

0f) 0)

nfn n

co) cO cn

I I

CEz

0.r- 0.CL 0.r

v-0.

buffer (50 mM

N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid [HEPES] adjusted topH 7.5 with NaOH, 0.1 mM EDTA, 0.5 mM dithiothreitol, 10% [wtlvol] glycerol, and 100 mMNaCl) in thepresenceof different cold

compet-itor DNAs. After incubation, the mixtures were passed

through nitrocellulose filter papers and washed with the

samebuffer. The bound DNAwaseluted from thefilter and

loaded onto a 5% native polyacrylamide gel. DNA was

visualized by autoradiography. Eachlanecontained 5 ,ulof in vitro-translated product, labeled

Oris,

and the indicated amountofcompetitor DNA.

(ii) Gel shift assay. The DNA binding ofOBPwith

wild-type and mutant origins wasanalyzed by gel shift analysis.

Bindingwascarriedoutby the method describedby Deb and Deb(3). For each bindingassay, about0.3pmol of synthetic

proteinwasused. Bindingassayswererepeatedtwice. Care

was takento use the same batch of proteinwhen different

base-pair-substitution mutants in the same context were

assayed for protein binding. The synthetic protein was

FIG. 7. Gel shift analyses ofpOR-Sl mutants. del 1-534 was

incubated withpOR-SlorpOR-Slmutantprobesasdescribedinthe

text. Mutations testedare shown above. (A) Autoradiogramofgel shiftanalysis.Notediminishedcomplex formationinpOR-Slbs-11

and loss ofcomplex formationinpOR-Slbs-9andpOR-Slbs-10. (B) Histogram showing levels of complex I formation ofpOR-Sl

mutantsrelativetopOR-Sl.

analyzed by an SDS-polyacrylamide gel electrophoresis as

described by Deb and Deb (3a). A single bandcorresponding to themolecular weight of del 1-534 wasobserved in each

case. For the DNAbinding, thein vitrotranslationmixture

was incubated with 32P-labeled wild-type or mutant

Oris

probe on ice for 30 min in a buffer composed of 50 mM

HEPESadjustedtopH 7.5 with NaOH, 0.1 mMEDTA,0.5 mMdithiothreitol, 10%(wt/vol) glycerol,and100 mM NaCl. Loading dye was added up to a volume of 10% of the

incubatedsample. The mixturewasloadedontoanative 5%

polyacrylamide gel made with 0.5x TBE (3a) and run at

roomtemperatureat30 mA. Thegelwasfixedinasolution

of 10% acetic acid and 2%glycerol, dried, and autoradio-graphed.

RESULTS

ReplicationanalysisofmutantsinsitesI, II,and III of

Oris.

To determine the role of sites I, II, and III in DNA

replication, we tested replication efficiencies of four

Oris

derivatives depicted in Fig. 2. del 1 deletes part of site I; pOR-Sl deletes site II; bs-15 substitutes three consecutive basepairs in siteIII;del31 deletesboth sitesIIandIII.Test plasmids were transfected into Vero cells, followed by

infection with HSV-1 (strain KOS). The cells were

har-vested, and DNAwas extracted at 16 hpostinfection. The isolated DNA wascutwithBamHI andDpnI andanalyzed by Southern blothybridizationasdescribed in Materialsand Methods. Replication analysis depicted in Fig. 2 shows that

noneof themutantsreplicatedasefficientlyasthewildtype. LongerexposuresindicatebothpOR-Sl and bs-15 replicate at10to 15%efficiencycompared with that of the wildtype. A number of different experiments (not shown) indicate a

decreased level of origin function for pOR-Sl and bs-15. Deletion in site Iordeletion in site IIaswellas site III has moredrastic effects onreplication. No significant difference

in replication efficiencies was observed previously (6)

be-tweenthe wildtypeandpOR-Sl with BHK-C21 cells anda

particular isolate of HSV (F). These experiments have been repeated several times with closer attention toquantitation

ofreplication data with the sensitive DpnIassayprocedure.

The importance of sites I, II, and III has also been

demon-strated by others (15, 28).

Replicationanalysis ofmutationsinsite Iof

Oris.

To have

a closer look at the functionally important nucleotides in

pX)R-S1

pOR-S1 bs-7

pOH-SI

bs-8

pOR-Sl bs-9

pOR-Si bs-10 pOR-Sl bs-l1

VIROL.

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PAUNDROME

III

I

11

5-CGGGT A AAGAAGTGAGAACGCG AGCGTTCGCACTTCGTC AATATATATATATTATT GGCGAAGTGCGAGCAC

GGCGCCGGCCCCGG-3_

3-GCCCAT TTCTTCACTCTTGCG trCGCAAGCGTGAAGCAGG TTATATATATA-AATAA CCGCTTCACGCTCGTG CGCGGCCGGGGCC-5

GGT bs-14

AGA bs-15

GAG bs-17

GCCCATTTTCTT AC2CTTGCGCTTCGCAAGCGTGAAGCAGGGTTATATATATATAATAATCCCGCTTCACGCTCGTGACCGCGGCCGGGGCC 16 del

A

GCAACTTTTCTTCACTCTTGCGCTTCGCAAGCGTG AGCAGGGTTATATATATATAATAATCCCGCTTCACGCTCGTGACCGCGGCCGGGGCC bs-18d(&

AAA A

A.

-o oo uI

-n

0L)

-C v

io

(o Ul)

.0v 0 .0

_-. :.-.m.jhhl -Progeny

N

Input

B.

1.0-0

c)

C)

c 0.5-0 CO C)

Is

_

L)f (D

.0

T- CL

>

U) (/) :

cn cn

-J n 3:

site I and site III, we generated a series of mutations

acrossthisregion.Thesewerethentested for DNA

replica-tionand OBPbinding. Mutant bs-7 resulted inadrastic loss

inDNAreplication (Fig. 3).Partof thismutation fallswithin the OBP footprint but is outside of the critical binding domain (3, 9, 18). The mutation introduced by bs-8 also markedly reduced DNA replication. This mutation falls completelywithin the OBPfootprintin site I (3, 10, 11, 18,

24) and is immediately adjacent to the critical binding domain. Mutants bs-9 and bs-11 had very low levels of replication. These mutants are within the critical OBP-bindingdomain(3, 18). Themost drastic loss inreplication

was seen withbs-10. This mutation isinthecentral part of the critical binding sequence. No replication signal was

noted, even in overexposed autoradiograms (not shown).

bs-12andbs-13 also hadverylow levels ofreplication. Thus,

theintegrityof thissetof residuesacrosssite I isabsolutely

critical in order to maintain an efficient level of DNA replication. The residues most crucial are represented by

mutants bs-9, bs-10, and bs-11. These correlate to the

FIG. 8. Replication of site III mutants. Wild-type and mutant origins were tested as previously described. Mutations tested are shown above.(A)Autoradiogram of replicationassay. Theposition ofthe progeny and input DNA is shown. Note loss in replication efficiency of mutant originsrelative to wild-type levels. (B) Histo-gram showing relative replication efficiencies of site III mutants versuslevelof wild-type Oris replication.

sequencespreviously shown to represent critical sequences for OBP binding at this site (3, 18). Residues outside the critical binding domain are also required for efficient DNA replication.

Figure 4 shows a histogram obtained after replication analysis of pOR-Sl mutants with mutations in the OBP-binding site I. None of the mutants replicated significantly. This is not unexpected because, even in the presence of site II, site I mutants have very low levels of origin acti-ity.

Gelshiftanalysis of OBP-binding siteImutants. Totestthe binding of OBPto mutantorigins, theDNA-binding domain of OBP, a C-terminal 318-amino-acid fragment, was gener-atedby usinginvitrotranscriptionand translation systems.

Figure5 demonstrates the sequencespecificity of bindingof

the wild type and del 1-534.Competitionbinding assays were carriedout withspecific (pOR-S) andnonspecific (pBR322) DNAsascompetitors. Theautoradiogramwasalso scanned by laser densitometer to quantitate the extent ofbinding.

Binding was inhibited more efficiently by pOR-S (2- to

10-fold) than by pBR322 (0- to 2.5-fold) under a 50 molar excessofcompetitoroverthe probe. This suggests that the DNA-binding observed is highly sequence specific. Addi-tionalobservationswithcold

Oris

mutantDNAsas compet-itors suggest that del 1-534 recognizes that same critical residues in site Ias does intact OBP(3b). Thisprotein (del 1-534) was usedfor gel shift analysis with 32P-labeled wild-typeandmutantoriginsasprobes.OBP haspreviouslybeen showntobindto atleasttwositeson

Oris,

site I and site II. SiteIhas been showntobe the strongestbindingsite(10). A deletion in site I abolished OBPbindingaswellasreplication

(3). To determine the roleofsiteI residues in OBPbinding,

substitutionmutants were tested for theirbindingefficiency

by using gel shift analysis. As shown in anearlier section,

these substitution mutants show a considerable loss in replication efficiency. The results of the gel shift analysis with the invitro-synthesized protein are showninFig.6.The

figureshowsformation oftwocomplexes,suggesting binding

at one (complex II) or both (complex I) sites. The binding experimentswererepeatedtwice. On eachoccasion, similar

patterns were observed, with about 10 to 15% variation in

binding when laser densitometry was done on the

autorad-iograms (datanotshown).

These resultsindicatethatsubstitutions within thecrucial

binding domainof OBP are detrimental to OBP

binding

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4366 MARTIN ET AL.

PAUNDROME

III I II

5'-CGGGTAIAAAGAAGTGAGAACGC AGCGTTCGCACTTCGTC AATATATATATATTATTAGCGAAGTGCGAGC ACGCGCCGGCCCCGG-3,wildt

3-GCCCAI1TTCTTCACTCTTGCG CGCAAGCGTGAAGCAG TTATATATATATAATAA CCGCTTCACGCTCGTG CGCGGCCGGGGCC-5 yp

GGT bs-14

AGA bs-15

GAG bs-17

GCCCATTTTCTT ACTCTTGCGCTTCC,CAAGCGTGAAGCAGGGTTATATATATATAATAATCCCGCTTCACGCTCGTGACCGCGGCCGGGGCC 16 del

A

GCAACTTTTCTTCACT-CTTGCGCTTCGCAAGCGTG AGCAGGGTTATATATATATAATAATCCCGCTTCACGCTCGTGACCGCGGCCGGGGCC bs-18del

A AA A

A. a,

o r-, )D I I +

co0

co~

.0

a)

OL

+1

0)

Q

>1

--I Protein

FIG. 9. Gel shift analysis of site III mutants. del 1-534 was incubated with wild-type or mutant Oris probe as previously de-scribed. Mutations tested are shown above. (A)Autoradiogram of gelshift analysis.(B)Histogramshowing levelsofcomplexIand II formationofmutantoriginsrelative towild-typeOris.

4 Complex

4 Complex 11

4 Free DNA

B.

*CoI1plcx

I

C(omnplex

II

10~

C

0 -.

0.5

-

;

-0 T

v

Trl

Tl

r-.0 Ir

substitutionmutantswere testedfor their abilitytoreplicate in thetransient in vivo replication assay. Figure 8 shows the results of this assay, alongwith ahistogramto comparethe relative replication efficiencies. Mutants bs-14 and bs-15 replicated poorly. These mutants map entirely outside of site I. Furthermore, they map to a region that by sequence similarity would represent the critical nucleotide sequence for the OBP recognition within site III. Interestingly, a

S.5

Site 5- AGCGTTCGCACTTCGTCC -3

Site 11 5'- AGtLGcTCGCACTTCGcCC -3!

Site ll 5- cGCGTTCtCACT TCtT t t -3

I. C.

Il >1

a) -D

U) BC

NOW .Progeny

<a) a)

-L >, >

a) V -o

Protein ri++

Cormiplex Ill E ..

Complex 1

-Complex 11 1 _

well as to DNA replication. The substitutions outside this crucial sequence (bs mutants 7, 8, 12, and 13) are more tolerant for OBP binding than for replication. This suggests a primary role of OBP binding for the residues inside the crucial domain. Residues outside the crucial domain, al-though essential for efficient replication, may not be directly involved in binding OBP. An involvement of these se-quences, however, in attaining the right structure for a preinitiation complex cannot be ruled out.

Todetermine the effects of the absence of siteII on OBP

binding to site I mutants, we used pOR-Sl derivatives as shown in Fig. 7. Clearly, the effect on OBP binding observed with these mutants is similar to that seen with site I mutants

havinganintact site II. Itseems, however, the reduction in

bindingcapability is more drastic when siteIIisabsent. This mayindicate cooperativity between the two sites as reported

elsewhere (2a, 9).

DNAreplication analysis of site IIImutants. To determine the role of site III residues in DNA replication, several

Free DNA F

UEUb -lnput

FIG. 10. Effect of an additional site I in Oris. (A) Sequence comparisonof sitesI, II,andIII. The overline indicatesthe critical OBP-bindingdomain identifiedearlier(3).SiteIsequenceis shown incapitalletters. SitesIIand III are from thestrand opposite to that used for siteI.Theyareshownthis way to demonstrate similarity of their nucleotide residues with those of site I.Connecting dots and capitallettersin sitesIIand IIIindicate identicalresiduesbetween thesites. (B)Replicationof site I-A. Mutant site I-A was constructed by usingcassette-directed mutagenesis. The site III sequence was replaced by a site I sequence in the context of the whole origin. This sequence, site I-A, is in the same relative orientation as site III. The replicationassay wasperformed as previously described. The posi-tions of progeny andinput bands are shown. Mutant site I-A did not replicateasefficientlyasthe wild-type origin. (C) Gel shift analysis of siteI-A.Thegelshift assay wasperformedaspreviously described. Note enhancedcomplex I andIIformation in the site I-A lane and the formationofathirdcomplex,III.

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Ons

III

I

II

CGGGTAAAAGAAGTGAGAACGCGAAGCGTTCGCACTTCGTCCCAATATATATATATTATTAGGGCGAAGTGCGAGCACTGGCGCCGG

attaTAAAAaAAGTGAGAACGCGAAGCGTTCGCACTTtGTCCtAATAatatatataTtaTtaGGacAAagtgcgaaCgCttCGCgtt

aGGGTAAAAGAAGTGAGAACGCGAAGCGTTCGCACTTCGTCCtAATAgtatatataTtaTtaGGgcAAagttGcGgCactGgCgCcc CttcTAAAAaAAGTGAGAACGCGAAGCGTTCGCACTTtGTCCtAATAgtatatataTtaTtaGGacAAagtgcgaaCgCttCGCgtt CctGcAccctgAtTGgcccaGaGgccCGTTCGCACcaatcaCCAATAagTtTtaATaATaAttattgcaacaaAGtgugaaCaCtac tcatgttttGgcaTGtGtcCaaccAcCGTTCGCACTTtcTttCtATATATATATATataTAtatatAtaTatatagAgaaagagaGa

HSV-1 oris

HSV-1 oriL

HSV-2 oris HSV-2 oriL

EHV ori

VZV ori

FIG. 11. Comparison ofOrissequence. The HSV-1 Orissequence was compared with severalherpesvirusDNAreplicationorigins to determine conserved residues. Nucleotides in HSV-1 OriL, HSV-2OrisandOriL,varicella-zoster virus (VZV)Ori, and an equine herpesvirus (EHV) origin identical to HSV-1 Oris are shown in capital letters. OBP-binding sites I and II and the presumptive siteIIIare shown for reference on HSV-1Oris.

single-base-pair deletion, represented by16del,alsoshowed a dramatic loss in DNA replication. bs-17 also had dimin-ished replication efficiency, although the level of replication was slightly higher than seen for bs-14, bs-15, and 16 del. One residueof this mutant (bs-17) falls into what may be, by sequencecomparison to site I, a critical sequence in site III. bs-18 del also had a low level of DNA replication. Thus, sequences in site III of

Oris

areessential for

Oris-mediated

DNAreplication.

Gel shiftanalysis of OBP bindingtositeIIImutants of

Oris.

Gel shift analyses were performed to test the ability of the site III mutants to interact with the DNA-binding domain of OBP. All of the mutants in this region of the origin had approximately the same level of complex II

forma-ion (Fig. 9). However, there was a difference seen in the

ability to bind the origin at two sites, since bs-14, bs 17, and bs-18 del all had lower levels of complex I

forma-ion. In bs-15, the ability to form complex I was actually enhanced to some extent. However, this mutant replicated

the least efficiently of all of the site III mutants. Thus, it seemsthatspecific mutations in the siteIIIregion markedly reduce the efficiency of DNA replication and alter overall OBP binding to the origin, even though these mutated sequences are well separated from defined OBP-binding

sites.

Analysis ofan

Oris

mutant with a second site I in placeof site III. No sequence-specific OBP binding has yet been demonstrated at site III, although we demonstrate above thatmutations in thisregionhaveaneffect on OBPbinding

to

Ori..

To determine whether strong OBP binding at the

position of site III would facilitate

Oris

replication, we constructed a mutant origin (site I-Amutant) that changed

site III into an exact copy of site I, while keeping the orientation of site III unchanged. We then tested this

origin

for theabilitytoreplicateandbind OBP. The resultsof these two assays are shown in Fig. 10. The site I-A mutant

replicated, butat alowerefficiency thandidthe wild type. Gel shift analysis revealed that bothcomplex I and IIwere present and were enhanced relative to wild-type levels. In

addition,athird complex(III), whichran more slowlythan

complex I, was apparent. Presumably, this is due to OBP

bindingatall three siteson the mutantorigin. Thus, strong OBPbindingtothethird site in thiscontextisdetrimentalto

originfunction.

DISCUSSION

Oris

derivatives with mutations in sites I, II,andIIIwere tested for DNAreplicationinaninvivo transient assay. Our

results (Fig. 2) show that mutations in any of these sites drastically reduce DNAreplication, signifying the functional importance of all three sites. The site I mutation seems to be themostdrastic. A decreased levelofDNAreplication was, however, observed with mutations in sites II and III. The

quantitativedifferences inreplication efficienciesof

Oris

and

Oris-1

reported here compared with those reported earlier

(6) may be due to improved assay conditions, including

rigorous quantitationof DNA before and aftertransfection,

in addition to the use ofdifferent strains of virus and cell lines.

Wegeneratedaseries ofspecificmutationsacrossthe

Oris

region spanning OBP-binding site I through a presumptive

thirdbindingsite(siteIII) both in the presence and absence

of site II. The exact nucleotide requirements have not been defined in this region. We first assayed the ability of

mutants across site I toreplicateandtobindOBP(Fig. 3, 4, 6, and7). Similarresults were obtained with thetwo series of mutants. bs-7 was found to result in a low level of

replication. Thismutantdoes notcompletelymapwithin the OBP footprint at site I (3, 10, 11, 18, 24). It falls directly

between site I and the A-T stretch. It is possible that the mutations introduced result in some structuralchange in the DNA thatdoesnotallowfor efficientinitiation. Inaprevious

paper, Doelberg and Deb (6) showed that a mutant

desig-nated bs-1, which mutates the first three base pairs of the A-T stretch near binding site I, also lost its replicative ability. They proposed that this site might be required for initiation of origin melting. Thus, it is possible that the mutation introduced by bs-7 may also be affectingasimilar function.

Mutantbs-8 falls within the OBP

footprint

(3, 10, 11, 18,

24). Thismutantshowsaloss in DNAreplication. Several of thesubstitution mutantsresideacross aregionwhose nucle-otide sequence elements were determined tobe critical for OBPbindingwithin thefootprint. bs-9 isan

example

ofone of these mutants. Here, we see that there is a direct correlation between a significant loss in OBP binding and DNA replication. Koffand Tegtmeyer (18) have

proposed

that the OBPrecognitionsite may berepresented

by

a setof

pentanucleotides, inverted relative to each other on the different strands

(5'-GTTCG-3'/3'-GCGTG-5')

with a

2-bp

overlap. The mutation introduced

by

bs-10 represents the most drastic mutation in this region, sincecrucial residues arechanged from both strands of the

overlap.

Thismutant did not replicate. No

signal

was detected even when the

autoradiogram was

overexposed.

Interestingly, this mutant had the lowest level of complex I formation. bs-11 is a

mutantthatresides within the

footprint

and

disrupts

a

single

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residue of thecritical

binding

domain identified earlier. This

mutantstill hasalow

replication efficiency,

even

though

the level of OBP

binding

is

slightly

increased. bs-13 isa

double-base-pair

substitution

mutation, essentially

atthe

junction

of

site I and site

III,

in whicha

single

residue is within the site

I

footprint

(3, 10, 11, 18, 24).

This mutant showed a slight

increase in

binding

relative to the other site I mutants but

replicated

poorly.

Base substitutions in the critical

binding

domain showacorrelation between

binding

and

replication.

Basesubstitutionsoutside the critical

binding

domain donot correlateaswell. These sequences may be

required

todirect

orientation of other

replication

factors or to allow for the formation of the correct structure ofa

nucleoprotein

com-plex.

A series of mutations were also created to allow us to determine critical residues in the site III

region.

The

repli-cation

capacity

and

OBP-binding capacity

of thesemutants

were

analyzed (Fig.

8 and

9).

In this context,

bs-14, bs-15,

and 16 del all had very low levels of

replication.

These mutantsreside ina

region

that

would, by

sequence

similar-ity,

represent the critical

binding

sequence as seenin site I

(3,

10, 11,

18,

24).

Mutantbs-14resulted ina

significant

loss

of

complex

Iformation. The central

question

remains,

since

no

binding

has yet been demonstratedtosite

III,

what is the

roleof theseresidues in

mediating

efficient DNA

replication

and overall OBP

binding

to the

origin?

We find it very

interesting

that

specific

substitutionsina

region

welloutside

of defined

OBP-binding

sites would have such a negative effect on

origin

function. There are several

possible

expla-nations for

this,

all of which are

speculative

at this time and will

require

further

analysis.

First,

it is

possible

that

OBP

actually recognizes

the site III sequence inan in vivo

setting.

This may be due to the presence of otherfactors

that

change

the overall structure of the

origin, thereby

making

the site more

accessible,

or to factors that

through

protein-protein

interactions allow OBP to sit at this site.

However,

our studies show that strong OBP

binding,

as

evidenced

by

studies withmutantsite

I-A,

is detrimentalto

origin

function.

Thus,

strong DNA

binding

alone cannot

explain

the functional role of these sequences. It is clear

from

previous

studies that the site III sequence alone is not

sufficient for

sequence-specific

OBP

binding

(9, 28). Thus,

any interaction of OBP with the

origin

at these sites must

be aided

by

the in vivo environment and is not seen in thecurrent conditions used for in vitro

DNA-binding

stud-ies.

Second,

it is also

possible

that OBP does not interact

with site III

directly.

Thus,

one can propose several func-tions that may be

provided

by

this

region.

Itis

possible

that these residues are

important

in order to

provide

sites for other factors to act upon as the

replicon

is formed. One

can envision OBP

binding

and the

generation

ofa

special-ized

nucleoprotein

structure whose

integrity

would be

cru-cial in order to

provide

for the

appropriate

interaction of other factors involved in

replication.

Any

mutations in these residues would

drastically

reduce the

efficiency

of the reaction. Within the same context, residues in site III may be

important

in the

secondary

structure of the origin.

It is

interesting

that

changes

in site III result in an overall

loss of

origin binding.

Thus,

the

secondary

structure of the

origin

may be

changed

such that OBP cannot interact at

wild-type

levels. This would then be manifested in the in

vivo loss of DNA

replication. Third,

thefar-left armofthe

Oris

region

contains ashort stretch which isA-Trich.This,

along

with the central A-T-rich

region,

may mediate a

conformationonthe

origin

that is

required

for efficient OBP

binding

and DNA

replication.

If this were the case, the

mutations should result inan alteration oforigin conforma-tion. Thismay manifest itselfby a loss in OBPbindingand DNAreplication.

The sequenceof the leftarmof the HSV-1

Oris

regionis well conserved among several other herpesvirus origins of DNA replication (Fig. 11). A comparison between HSV-1

Oris

and

OriL

(27, 29), HSV-2

Oris (30)

and

OriL

(19),

varicella-zoster virus Ori (26), and the equine herpesvirus

Ori (1) demonstrates a region of high conservation that corresponds to the critical sequences required for OBP binding at site I. This 9-bp region, 5'-CGTTCGCAC-3', is conserved in alloriginscompared. This conservation

corre-lates well withourresults, since these residueswerecritical fororigin function. Other residues throughout the leftarm

arealso well conserved. A total of 10 other residues

through-out were present in four or more of the origins analyzed.

Thus, it is expected that these residues represent crucial functionalelements in theseregions. Mutagenesis of site III would disrupt the integrity of these residues and result in diminishedorigin function. Thiswasthecaseinourstudies. The far-left arm containsa stretch in which 7 of 8 residues are A orT. These residues may mediate the adoption ofa conformation important for origin function. This general

motif is also maintained in these virus origins. In the varicella-zoster virus origin, although there is not aperfect sequence match, the general A-T-rich character is main-tained at this site. This may represent a common element necessary for efficientoriginfunction.

We have createdspecific mutationsacrossthe left armof the HSV-1On5 regionin ordertoclearly define theelements requiredfor efficientinitiation of DNA replication andOBP binding. Our results demonstrate the need for residues in site I and site III to mediate efficient DNA replication. Residues in thecritical OBP-binding domainarerequired for bothDNAreplication and OBP binding. However, residues outside this domain, although notcrucial for OBP binding,

are necessary for DNA replication. This suggests that the binding of the origin sequence by OBP is not the only determining factor for the initiation of DNA replication. Single-base-pair mutagenesisis requiredto more finely dis-sect the intricacies oforigin function. Our results demon-stratethe crucialnatureofseveral groups of residues within the origin and begin to implyafunction mediated by these sites.

ACKNOWLEDGMENTS

This workwas supported by grants from the American Cancer Society (MV-420), the Elsa U. Pardee Foundation, andthe Basil O'Conner Starter Scholar Research Award from the March of DimestoSumitraDeb. This work is also supported by anAmerican Cancer Society Institutional Grantto SumitraDeb and an Institu-tional Research Grant to Swati Palit Deb. David W. Martin is supported in part by a NIH predoctoral fellowship in microbial pathogenesis.

Wethank Jane Tatefor excellent typing assistance.

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