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0022-538X/93/042123-10$02.00/0

Copyright © 1993, American SocietyforMicrobiology

Construction and

Properties

of

a

Recombinant Herpes Simplex

Virus 1

Lacking

Both

S-Component

Origins of

DNA

Synthesis

KAZUHIKO IGARASHI, RANDALLFAWL, RICHARDJ. ROLLER,ANDBERNARD ROIZMAN* TheMarjorie B. KovlerViralOncology Laboratories, The University of Chicago, Chicago, Illinois60637

Received 11 September 1992/Accepted 23 December 1992

Theherpes simplex virus 1 (HSV-1)genomecontains three origins of DNA synthesis (Ori)utilizedby viral DNAsynthesis proteins.Onesequence

(OriL)

mapsin the Lcomponent,whereastwosequences

(Oris)

mapin

the Scomponent.Wereportthe construction ofarecombinantvirus, R7711, from which both

Oris

sequences

have beendeleted, andshowthat the

Oris

sequences arenotessentialforthereplication of HSV-1 in cultured cells. In addition tothedeletions of

Oris

inR7711, the a47 geneand the 5' untranscribed and transcribed

noncoding regions of the

Us11

gene weredeleted,oneof the a4promoter-regulatoryregionswasreplaced with

thesimianvirus40promoter,and the at22promoterwassubstituted withtheav27promoter.The totalamount

ofviral DNAsynthesizedinVero cells infected with the

Oris-negative (Oris-)

viruswasapproximatelythat

seenincells infectedwith the

Oris-positive

virus. However, cells infectedwith the

Oris-

virus accumulated

viralDNAmoreslowly than those infected with the wild-type virus during the first few hoursafter theonset of DNA synthesis. Insingle-step growth experiments, the yield of

Orins

progeny virus wasreduced at most fourfold.Althoughasingle

Oris

(R. Longnecker and B. Roizman, J. Virol. 58:583-591, 1986) and the single

OriL (M. Polvino-Bodnar,P.K.Orberg, andP.A.Schaffer, J. Virol.61:3528-3535, 1987) havebeenshown tobedispensable, this isthe first indicationthatboth copiesof

Oris

aredispensable and thatonecopyofan

Orisequencemaysuffice forthereplication ofHSV-1.

The herpes simplex virus 1 (HSV-1) genome consists of two covalently linked components, L and S, that are

in-verted relativetoeachothersuchthat DNA extractedfrom virions consists of four populations differing solely in the relative orientations of the components (7, 11). Each com-ponentconsists ofauniquesequence(ULor

Us)

flankedby

inverted repeats (31, 38). In addition, each component contains either one (OriL; L component) or two

(Oris;

S component)sequencesthatconferuponDNAfragmentsthe capacity tobe amplified by proteins specified by thevirus (15, 21, 33, 34, 35, 37, 41). The OriL sequence maps

approximatelyin the middleof theuniquesequenceof the L component (UL), whereas the two

Oris

sequences map

within therepeatsflanking the uniquesequence

(Us)

ofthe S

component. BothOriLand

Oris

are locatedbetween diver-gentlytranscribedgenes; OriLislocated betweenthe UL29

and UL30 genes, whereas

Oris

maps between the a4gene

andeither the a22orthea47gene. Thetwo

Oris

sequences areidentical but inverted relative toeach other.Moreover, they share homologous sequences with the OriL sequence

(15, 41).The evidence that both

Oris

sequencesand the

OriL

sequence can function as aDNAreplication origin utilized

byHSV DNA replication proteins has been obtained in a

transientplasmid replication assay (15, 35, 37, 41). Also, a

protein specified bythe UL9 open reading frame has been showntobindtospecificsiteswithin eachorigin (9,23, 40), and the UL9gene isessential for the replication of HSV-1 (4). Nevertheless, mysteries regardingthe functions of the originsof DNAsynthesis duringviralreplication abound.

Akey problem stemsfrom thenatureof thereplicationof HSV DNA. This DNAcircularizes withinaveryshort time after infection inthe absence of denovoprotein synthesis

(24). In principle, circular DNA could replicate by a theta model,whichdepends heavilyoneach initiation eventinthe

*Correspondingauthor.

origins ofDNA synthesis, or by a rolling-circle model, in

whichthedependence ontheorigins is minimal and occurs at most only once at the initiation of DNA synthesis. Attemptstouncoverforms consistentwiththeta replication have not been successful. The evidence is consistent with the conclusion that both defective genomes, amplified in trans by viral enzymes furnished by the expression of nondefective genomes, and nondefective genomes them-selves accumulate as head-to-tail concatemers, in

accor-dance witharolling-circle modelofDNA replication (3, 13,

14, 37). If this model iscorrect, itisnotclear why theHSV

genomecontainsandpresumablyconservesthreeoriginsof

DNAreplication.

Inpreviousstudies,workers inourlaboratory and

subse-quently others (16, 25) reported that at least one origin, eitherone

Oris

orOriL,canbedeletedwithoutaffectingthe

ability of the virus to replicate in cells in culture.

Further-more, OriLisnotrequiredfortheestablishment and

reacti-vation of the virus in trigeminal neurons harboring latent

virusinmice inoculatedbythecornealroute(25).Attempts todeleteboth

Oris

sequencesfromthe HSV-2genome were

unsuccessful,andit has beensuggestedthatatleastone

Oris

sequenceis essential for viral DNAreplication (32).In this

paper, we reportthe properties ofagenetically engineered

virusfromwhich both

Oris

sequencehave been deleted. The phenotypic properties of the recombinant cannotbe differ-entiated from those of the wild-typeparentwith respectto DNAsynthesisin cells in culture.

MATERIALSAND METHODS

Cells and viruses. The properties and propagation of HSV-1 strain F [HSV-1(F)] and the thymidine kinase (tk) deletion mutant HSV-1(F)A305 were previously described (8, 26). Vero cells (American Type Culture Collection), rabbit skin cells (obtained fromJ. McClaren), and 143TK-2123

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2124 IGARASHI ET AL.

cells (obtained from Carlo Croce) were propagated as

de-scribed elsewhere (2).

Plasmids.Standard procedureswere usedinall

construc-tions described in this report (29). Plasmid pRB124 carries theBamHIXfragmentofHSV-1(F)inpBR322(16).pRB179 carries the BamHI Tfragmentof

HSV-1(F)

in

pGEM-3Z

(1).

Thesequencearrangementofplasmidscarrying thea4gene

fusedtothepromoter-enhancer sequenceofsimianvirus 40

(SV40) is shown in Fig. 1. Plasmid pRB4325, in which the HSV-1 tk gene fused to the ao27 promoter-regulatory

se-quencesisinserted into the a22

promoter-regulatory

region,

was constructed in several steps. Inthe first step, a Hinfl fragment ofpRB257 containingnucleotides -968to +53 of the a27 gene was cloned into the NruI site located down-stream of theao47promoter in pRB421 to generate plasmid pRB4012. The orientation of the ct27 promoter inserted in pRB4012was the same asthatof the a47promoter. In the second step, plasmid pRB4012 was partially digestedwith

TthlllI, filledin with the Klenow fragment ofEschenchia coli DNA polymerase, and ligatedwith a1.5-kbBglII-NcoI

fragmentencodingthetkgene,which had beenexcisedfrom

pRB3351 and filled in with the Klenow fragment. The resulting plasmid,pRB4276, carries thetkgeneclonedinthe

TthlllI site of the xt27 promoter-regulatory domain, in a

direction suchthat the tk gene is transcribed from the a47 promoter. In the final step, pRB138, which carries the BamHINfragmentofHSV-1(F) DNA in the BamHI siteof pBR322, was digested withRsrII, and the resulting larger fragment, which contains the

at22

coding sequences, was

ligated with the 2.7-kbRsrII fragmentofpRB4276to

gener-atepRB4325. The direction of ligationwassuch that the tk

gene wasunderthe control of theao22promoter,whereasthe

a22 gene was under the control of the ot27 promoter. For generation of pRB4397, inwhich the functional tkgene and

Oris

sequences are deleted, pRB4325 was digested with

BssHII, andthe larger fragmentwasrecircularized with T4

DNA ligase.Two DNA fragments withdeletionsacross

Oris

in the R7711 virus (see below) were cloned from the viral

DNA as BamHI fragments into pGEM-3Zf(+) (Promega, Madison, Wis.). pRB4488 carries the 1.3-kb BamHI frag-ment extending from theat4 5' transcribed nontranslated leader regiontothe BamHI sitepresentin the

at27

promoter-regulatory sequence. pRB4489 carries the 2.7-kb BamHI

fragment,whichincludes theSV40 promoter-ot4 fusion junc-tion.

Viral DNA. Vero cells in 850-cm2 roller bottles were

infected withvirusat amultiplicityofinfection of 0.01PFU percell and incubatedfor3to 4days at34°C. Forharvest,

the cultures were washed once with phosphate-buffered

saline (PBS) (140mM NaCl,3 mM KCl, 10 mM Na2HPO4,

1.5 mM KH2PO4), scraped into PBS, and recovered by

centrifugation at 3,000rpm in aBeckman JS-13 rotor for 5

min at 4°C. The cell pellet was resuspended in 150 mM

NaCl-1.5 mM MgCl2-0.1% Nonidet P-40-10 mM Tris-HCl

(pH 7.4), the suspension was vortexed briefly, and then

nucleiwereremoved bycentrifugation as described above.

Thesupernatantfluidwasremovedto afresh tube,adjusted

to0.2% sodiumdodecylsulfate-5mMEDTA-50 mM

2-mer-captoethanol,and extractedtwiceby gentleshaking with1:1

phenol-chloroform. Nucleic acids were precipitated with 2

volumesof ethanol andrecoveredby centrifugationat10,000 rpm in a Sorval SS-34rotorfor 15min at4°C. Thenucleic

acid pelletwas suspended in 1 ml of TE (1 mM EDTA, 10

mMTris-HCl [pH8.0]) andtreatedwith RNaseA(20

,ug/ml)

for 15min at 37°C.Viral DNAwas separated from

oligori-bonucleotides and other small contaminantsby

sedimenta-tion through a gradient of 5 to 20% potassium acetate in 10 mM

Tris-HCI

(pH 8.0)-5 mM EDTA at 27,000 rpm in a Beckman

SW41

rotor for 17 h at4°C. The supernatant fluid wasdiscarded, and the viral DNA pellet was resuspended in TE. Excess salt was removed by precipitation of the viral DNA with ethanol. The precipitate was resuspended in TE andstored at 4°C.

Recombinant viruses. Procedures for the construction of

recombinant HSVs by insertion and deletion of the tk selectable marker were previously described (27, 28). The structure and purity of recombinant viruses were evaluated by hybridization of diagnostic probes with restriction

en-zyme-digested DNAs from the recombinant viruses. Sam-ples of DNA were digested with restriction enzymes,

elec-trophoretically separated on 0.8% agarose gels, and

transferredtoZetaProbe membranes (Bio-Rad Laboratories, Richmond, Calif.). Blots were probed with 32P-labeled DNA by use of protocols recommended by the manufacturer.

Double-stranded DNAs to be used as probes were labeled with

[a-32P]deoxynucleotide

triphosphates by use of a nick

translation kit (DuPont, Wilmington, Del.). A synthetic DNA

oligonucleotide

probe was labeled at its 5' terminus with

[-y-

P]ATP and T4 polynucleotide kinase.

Transient replication assay. Test plasmids (0.5 ,ug) were

transfected into Vero cells grown in 25-cm2 flasks by the calcium precipitation method (29) with 4.5 ,ug of salmon sperm DNA as a carrier. Four hours later, the cells were incubated with 15% glycerol in PBS for 2

min,

washed with PBS without glycerol, and reincubated at 37°C. At 19 h

posttransfection, the cells were exposed to 10 PFU of HSV-1(F) per cell for 1 h. At 32 h posttransfection, total cell DNA was extracted from the cells as described elsewhere (5). Portions of theresulting DNA samples equivalent to 106 cells were digested with 40 U of BamHI and 100 U of DpnI overnight. The digests were separated on 0.8% agarose gels,

transferred to ZetaProbe membranes, and hybridized with

nick-translated pGEM-3Zf(+) DNA.

Time course of viral DNA synthesis. Vero cells in 25-cm2 culture flasks wereinfected with 5 PFU of test virus per cell. Atvarious times afterinfection, total cell DNA was isolated asdescribed elsewhere (5), and one-fifth of each sample was digested withBamHI.Digests were electrophoretically sep-arated on agarose gels, transferred to ZetaProbe mem-branes, andhybridized successivelywith excess 32P-labeled pRB179 and pRB124 DNAs carrying BamHI T and BamHI XfragmentsofHSV-1(F) DNA, respectively. The amount of

radioactive DNA hybridizing to each band was quantified with aBetascope 603 blot analyzer (Betagen).

RESULTS

Experimental design. To determine whether HSV-1 re-quired at least one

Oris

sequence,wesystematically deleted both

Oris

sequences. Thestrategy used in theseexperiments

was as described earlier (27, 28) and was based on a technique in which the tk gene selectable marker, sand-wiched between flanking sequences homologous to the tar-get sequence, was first recombinedat a site at or near the target sequence and then deleted along with the target sequence in a second recombinational event. The proce-dures were straightforward and relied on the observation thatalthough double recombinational events through flank-ing sequences are relativelyinfrequent, procedures for the selection of either tk-positive ortk-negative virus progeny resulting from such events are available (27, 28).

Theinitialstrategy was to insert one copy of the tk gene J. VIROL.

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VOL. 67, 1993

A Plasmids

~~~~~L

U1

---_--- -' _

Oris

r rs

I

'v47~~~~IOrs

w22_-A4

0 _ i 4 a47 (

a40

kI ( 2

H NEBaS Ba Na E S BaBst N S

HSV-1

Oris-

VIRUS 2125 B.Viruses

I

usU

UL

TK

=:

[

TK -o [

,, -...--, -I k

S Ba Bst P/NE Ba S Ba

Ps

Nd P

Ons

a47 *40

a4 Us51 _- I

sv40

Us1o( _I

II A Ii 3

H/S Ba NaE S BaBst E (E)

d a bc

a -'L- - d _ __

-a d 'c '

OnS

Psv4Oa4

IS a S A

S Ba Na E (E)

Bst/Nd

Ons

U51O0_l

F-Psv40a4

11

I A

7r7k

S Ba/PH/S Ba NOE EBaS

Bst/Nd

Us5°

(

H_

Psv4Oa4

I S Ba/P H/S

- -Bst/Nd

- a4

II

Ba Na

- a4

Ba Na

4

Ba Sp P H/S Ba

i

i2

3

6 FIG. 1. Schematic construction of recombinant virus R3914. (A) Plasmid construction. Line 1,prototype sequence arrangementof the HSV-1genomeshowing uniquesequencesof the Land Scomponents(thin lines) flanked by invertedrepeatsequences(open rectangles). Line 2, expanded representation of the left and right ends of the Scomponent, showing the organization ofsequencescontained within plasmids pRB3094andpRB421, respectively. Transcribedsequences areindicatedbyarrows,protein codingsequences areindicatedby filled rectangles,

and Orissequences areindicatedbyopen rectangles. Line 3, right,pRB421was alteredbyendfillingof the unique EcoRI site (shownin

parentheses)andbyinsertion ofanEcoRIlinkerinto the NruI siteto createplasmid pRB441. To simplify the presentation of the construction, inthisdiagram ofpRB441,wedesignatedthesequencesbetween theSalIand BstEII sites with thelettera,those between the BstEII site and the EcoRI insert with the letterb,and those between the EcoRI insert and the end-filled EcoRI site with the letterc.Line3, left,thesequences

betweentheSall and HindIII sites ofpRB3094werereplacedwith the HindIII-NdeI fragmentofpSV2CAT (10),which contains theSV40 enhancer-promotersequencesfrom PvuIItoHindIII(hatched rectangle)and thepBR322sequencesfrom NdeItoPvuII(open rectangle).The resulting plasmid, pRB3906,had the a4 leadercodingsequencesunder the control of theSV40promoter(Psv4O);this chimericconstructcould be excisedas anNdeI-EcoRIfragment, designated bythe letter d. Line4,the bsequencesofpRB441werereplacedwith the dsequencesof pRB3906,sothat thesegments ofpRB441designatedaandcwouldprovide flankingsequencesfor the Psv4O-a4construct(inthe ordera-d-c) within theresulting plasmid, pRB3915. (B)Virus construction. Line1,DNAsequencearrangementofHSV-1 recombinant R3630(19) containing deletions in the naturalpositionof the tkgeneand inthe Scomponent. Line2, expanded representationof the Scomponent (right end)of recombinant virusR3630,in which the a4-tk chimericgenehasreplacedthecodingsequencesbetween theBstEIIandNruIsites of thea47gene. Transcribedsequences, protein coding sequences, and Oris sequences areindicated as inpanel A; a4 promoter-regulatorysequences are

depicted byastippled rectangle;and the tkgene5' transcribednoncodingandcodingsequencesareindicatedbyacross-hatchedrectangle.Line 3, diagram showingthe intendedreplacementof the a4-tkchimericgeneinR3630 with thechimericPsv4O-a4construct inplasmid pRB3915 by recombination within theregionsofUs10andOris.Line4,schematicrepresentationof the Scomponent (right end)ofR3914,withadeletion ofOrisresulting from recombinationwithin the regionsofUs10 and a4. Line5, expansion of the mutagenized regionin R3914,which is contained withina2.7-kb novelBamHIfragment.Line6,three restrictionfragments,whichtogetherspanthemutagenized regioninR3914,were usedasprobesinFig.2. Probe1,sequencesbetween the left BamHI site and the nearestSphIsite in the HSV-1BamHIZfragment; probe2, SV40 promoter-enhancersequencesbetween thePvuII and HindIII sitesexcised frompSV2CAT; probe 3,entireHSV-1BamHIYfragment. Restrictionenzymes:Ba, BamHI; Bst, BstEII; E, EcoRI; H,HindIII;N, NruI; Na,Narl;Nd, NdeI;P, PvuII; S,Sall;Sp, SphI.Note that at thejunctionindicatedasH/S,theHindIII site but not theSallsitewasregenerated.

adjacent to each of the two

Oris

sequences within the

invertedrepeats ofthe Scomponent,i.e.,between

Oris

and

the a22 gene in one repeat and between Oris and the a47 gene in the other. However, because such a recombinant

could not be selected in a single step, we resorted to

sequential mutagenesis of the

Oris

sequences.

Construction of HSV-1 lacking one copyof

Oris.

During construction ofavirus in whichachimeric cx4 gene

consist-ing of the coding domain of the gene fused to the SV40

promoterwasrecombined into theviralgenome, a recombi-nant virusdesignatedR3914 andlackingonecopyof the

Oris

sequencewas isolated. Theparentvirus used for this

con-_Wa4

2 Na

3

4

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2126 IGARASHI ET AL.

.-I

N cr

l- 11

o'

F.--4.

C

cn -gr cc =

T)

if

C) r-- N lce CN

_4_ -* b

- -4

40

-S

[image:4.612.158.470.76.339.2]

1 2 3 4 1 2 3 4 5 6 1 2 3 4 1 2 3

FIG. 2. Autoradiographic imagesofelectrophoretically separated digestsofwild-typeand recombinantvirusesprobedforstructure.(A) BamHI digests ofR7710(lanes1 and3)and R3914(lanes2 and4)DNAsprobedfirst with32P-labeledpRB138DNA(lanes1 and2),which contains theHSV-1(F) BamHIN DNAfragmentinpBR322,stripped,andreprobedwith32P-labeledpRB4540DNA(lanes3 and4),which contains theBglII-NruIDNAfragmentof the HSV-1 tk gene(20).Bandshybridizingtothe HSV-1sequencesareindicatedwith dots.The band marked with the arrowhead is the 2.7-kbfragmentcontaining the SV40promoter-a4 chimericgeneandpBR322sequenceshybridizing with theprobes (Fig.1B,line4). (B)BamHI digests ofR3914(lanes1 and4),R7710(lanes2and5),andR7711(lanes3 and6)DNAsprobed firstwith the50-meroligodeoxynucleotide probeshown inFig. 3, line2(lanes4to6), andthenreprobed with the 1.6-kb BamHI-SacI DNA fragmentofpRB138,which contains sequences betweenthe leftBamHI site and theSaclsite of the a22 gene,includingOris(lanes1to3). (C)BamHIdigests ofHSV-1(F)(lane 1),R3914(lane 2),R7710(lane 3),and R7711 (lane4)DNAsprobedwiththe 32-mer oligodeoxynu-cleotideprobeshown inFig. 3,line 3.(D)BamHI-BssHIIdigestsof R3914(lane 1),R7710(lane 2),and R7711(lane 3)DNAsprobedwith thesame 1.6-kbBamHI-SacI DNAfragment ofpRB138asthat usedinpanelB. Thepositions of markerDNAsareindicatedtotheleftof eachblot;their sizesare8.5,7.2, 6.4, 5.7, 4.8, 4.3, 3.7, 2.3, and1.9 kb. Inpanel D,thepositions of 1.4-, 1.3-, and 0.7-kb bandsarealsoshown.

struction, R3630 (19), carried an a(4 promoter-tk chimeric gene in place of the a47 gene (Fig. 1B, line 2). DNA of plasmid pRB3915 (Fig. 1A, line 4) was cotransfected with R3630DNA toisolateatk-negative virus. The possible viral DNA structures within the mutagenized region resulting from the double recombinational event in which the tk gene was deleted are depicted in lines 3 and 4 of Fig. 1B. If the

right-handcrossoverrelative to the SV40 promoter occurred within the region near

Oris,

the progeny virus would acquire the structure depicted in line 3, in which a total of three copies of the ot4 gene would be present in the viral genome and one copyof the a4 gene would be fused to the SV40 promoter. Ifthe right-hand crossover relative to the SV40 promoter occurredwithin the a4 gene domain, the progeny virus would acquire the structure depicted in line 4, in which oneoftwocopies of the a4 gene would be fused to theSV40 promoter and one of the

Oris

sequences would be deleted. On thebasisoftestsdefined by two criteria (see below), we concluded that isolate R3914 has the structure depicted in

Fig. 1B, line 4.

Thefirstcriterion was the presence or absence of a 3.5-kb BamHI fragment that contained

Oris

and the 3' portion of thea4gene(Fig. 1B, line 3). As shown in Fig. 2C, lane 2, the

diagnostic 3.5-kb fragment was not detected by

hybridiza-tion of the BamHI digest of R3914 DNA withalabeled DNA probe specific for

Oris

andshown inFig. 3.

The second criterion was the location ofanSV40 promot-er-a4 chimericgenein the DNAfragments generated bySall

digestion. If the recombinationyielded theconstructshown

AwT

I

/

5'-GGGTAAAAGAAGTGAGAACGCGAAGCGTTCGCACTTCGTCCCAATATATA-3'

FIG. 3. Diagramof the structureofOris and sequences of the oligodeoxynucleotides thatwereused as probes forOrissequences. Line 1, schematic structure of Oris. Rectangles labeled I to III representUL9 protein binding sites(IandII)or a presumedbinding site(III) (9, 22, 39). A/Trepresents an AlT-richsequence. Lines2 and 3 show the synthetic oligodeoxynucleotides that served as probesforOrissequences inviral DNAs in the studies shown inFig. 2. The50-merand 32-mer sequences show80% (40of50)and97% (31 of 32) identitytoOriL,respectively.

J. VIROL.

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BamHI BamHl BamHl/HindIll

W. WA_.f

_l% 01,M r=

;X - , X, v X,

BamHl BamHl/Hindill

P" I.I

_4

f- -,

o.-

r-II0L;E

'S , uS

~u :

1 2 3

SV40

4 5 6

SV40

7 8 9 10 11 12

BamHI/SphI

13 14 15 16 17 18 BamHI Y

ofBamlit Z

FIG. 4. Autoradiographic images of analyses of theSV40 promoter-a4 chimericgenein the R3914genome.Shownareautoradiographic

imagesofSalldigests (lanes1to3),BamHIdigests (lanes4to6, 7to9, and 13to15),orBamHI-HindIII digests (lanes 10to12and 16to 18)ofHSV-1(F)A305 (lanes 1, 4, 7, 10, 13, and16), R3630 (lanes 2, 5, 8, 11, 14, and 17), orR3914(lanes 3, 6, 9, 12, 15, and 18) DNAs

hybridizedwiththe radiolabeledprobes shown inFig. 1B, line 6. Two of the electrophoretically separated digests (lanes 1to3 and 4to6)

wereprobedwith the32P-labeledSV40 promoter-enhancer DNA. The electrophoretically separated digest shown in lanes 7to12wasfirst probedwith the32P-labeled0.29-kbBamHI-SphI fragment containing portions of theUs10andUs11genesoftheBamHI Z fragment. The probewasthenstripped off,and thedigestwasreprobedwith the32P-labeled BamHIYfragment (lanes 13to18). Note that the sizes of the BamHI Y and BamHI ZfragmentsofHSV-1(F)areroughly equaland that therearetwocopiesof the BamHI YfragmentinHSV-1(F) DNA, inasmuchasthefragmentmapsin the inverted repeatsequencesflankingtheScomponentinHSV-1(F). One BamHI Ysequence wasfused totheSV40 promoter-enhancersequencein R3914.Thepositionsof marker DNAsareindicatedtothe left ofeachblot;theirsizesare8.5,

7.2, 6.4,5.7, 4.8, 4.3, 3.7, 2.3, 1.9, 1.4, and 1.3 kb(the1.4- and 1.3-kbbandsarenotshownonthe leftmostblot).

in line 3 of Fig. 1B, the SV40 promoter-a4 chimeric gene

should be detectedby hybridizationwith theSV40promoter DNAas asingle SalIband 7.4 kb longbecause therewould be two SalI sites surrounding the SV40 promoter-a4 chi-mericgene.On the otherhand, if the recombination resulted in the construct depicted in line 4 of Fig. 1B, the SV40 promoter-a4chimericgeneshould be detectedasSallbands 13 and 7.5 kb long because of the isomerization of the S component, inasmuchas there would beonlyoneSall site upstream of the chimericgene. As shown inFig. 4, lane3, SV40 promoter sequences hybridized to two R3914 SalI DNAfragments of theexpected sizes. This result is consis-tent with the recombination event depicted in line 4 of

Fig. 1B.

Hybridization studies (Fig. 4) provided additional verifi-cation ofthe structure of the SV40 promoter-a4 chimeric geneinthe R3914DNA.Electrophoretically separated HSV-1(F)A305, R3630, and R3914 viral DNAs digested with

BamHI or BamHI-HindIII were hybridized with various

radioisotopicallylabeled DNAprobes.Thekeyresultswere

thata2.7-kbBamHIfragmentof R3914 thathybridizedwith a DNAfragment from the SV40 promoter-enhancer region (lane 6) also hybridized with the unique sequence of the

BamHI Zfragment (lane 9)and withtheBamHIYfragment

(lane15).Thepresenceof theHindIIIsitebetween the SV40

sequenceand the BamHI Ysequenceonthis2.7-kb BamHI

fragment (Fig. 1B, line 4) was confirmed becauseHindIII

digestedthis 2.7-kbfragmentinto 1.0- and 1.7-kb fragments that hybridized with BamHI Z (lane 12) and Y (lane 18) probes, respectively. These observations were consistent

with a proper fusion between the SV40promoter and one

copyof thea4gene.

On the basis of theseresults,weconcluded thefollowing:

(i)the sequences between the BstEII site of the BamHI Z

fragmentand theSalIsite of the BamHI YfragmentinR3914

werereplacedwith the SV40 promoter-enhancersequences

andaportionof thepBR322sequences; (ii) thecopyof the

a4 promoter-regulatory region nearest the a47 gene was

replacedwith theSV40 promoter-enhancer sequences; and (iii)the

Oris

sequencediscussed ingreaterdetailbelow,the

a47gene, and the5' untranscribed and transcribed

noncod-ingsequencesof the

Usll

genewere deleted.

Theviability of R3914 was consistent with theprevious observations thatonecopyof

Oris

could be deleted fromthe

virus genome withoutabolishing the ability of the virus to

multiply (16)and that theaL47andUs11geneswere

dispens-able forgrowth incell culture (17, 19).

Construction of HSV-1 lacking both copies of Ori5. In Sall

c T

xS

-- E

40

a

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2128 IGARASHI ET AL.

Jl UL us

,_____ ______---@, _

Oris _- IO°fiO

a4 V - a22 a0-4u7a4

Ba S Ba Bss N Ba Ba BaBstNBss Ba S Ba

BamHiN BamHIZ

USIO a a4

Ba S BaBss N Ba Ba Psv4O Ba

A'

Oris TK

a4 _1W- 0HOW' _ a22

ab a

Ba SBaBss Baa Ba Ba

TKPa27

a4 in-4 I-*. b a

Ba SBaBssBa

Pa27

L 11.J

[image:6.612.52.294.56.280.2]

Ba

FIG. 5. Summary ofgenomestructuresof recombinantviruses.

Line 1, schematic representation of the prototype HSV-1 genome

arrangement. Line 2,sequenceorganization of theBamHI Nand Z

fragmentsatthe left and right endsof the uniqueshortcomponentof the

HSV-1

genome, respectively. Transcribed sequences are

indi-catedby arrows, protein codingsequences are indicated by filled

rectangles, and Oris sequences are indicated by open rectangles. The positions of the BamHI N and Z fragments of HSV-1 are

indicated by the double-headed arrows. Line 3, corresponding

sequenceorganizationinrecombinant R3914.Thesequencelocated

between the BstEII and

SaII

sites ofHSV-1(F)A305,shown inline2,

which includesmostofthe BamHI Zfragment, wasreplacedwith

theSV40 enhancer-promoterfragment (rectangleb)andNdeI-PvuII fragment of pBR322 (rectangle a). Theunique 2.7-kb BamHI

frag-mentspanning thismutation (line A)wascloned from theprogeny

virusR7711,giving risetopRB4489. Line 4,same asline 2, except

that the structure of virus R7710 is shown. Rectangle b, tk gene

coding sequence; rectangle a,a27 promoter-regulatory sequence.

Line 5, same as line 3, except that the structure of recombinant

R7711 is shown. The BamHI DNA fragment that contained the

junction of the deletion(line B)was cloned into pGEM-3Zf(+) to yieldpRB4488.Restrictionenzymes:Ba, BamHI; Bss, BssHII;Bst,

BstEII; N,NruI;S,SalIl.

wild-typeHSV-1,

Oris

is located in the promoter-regulatory

sequencesof both the

at22

and thea47 genes(Fig. 5, line 2).

In making insertions and deletions around the remaining

copy of

Oris

in R3914, we wanted to ensure continued

expression of theaL22gene, since this gene isimportant for growth in primary human and rodent cells (30)and sincethe properties ofadouble deletion ofao47and a22 could not be predicted. To accomplish this, we created twoplasmids: (i) pRB4325 (Fig. 5, line 4), in which both the tk selectable marker and the promoter-regulatory sequences of the

(x27

gene were insertednear

Oris

in thea22genein the BamHI

N fragment such that thetk gene wasunder thecontrol of the

at22

promoter, whereas the a22 gene wasunder the control

of theaL27 promoter, and (ii) pRB4397 (Fig. 5, line 5), in which

Oris,

the

aL22

promoter-regulatorysequences, and the

tk

selectable markerweredeleted. The deletion in pRB4397

didnotremove anyTAATGARATmotifs, important fora4

gene expression, from the region upstream of the a4 gene

promoter. The

tk

insertion plasmid pRB4325 was

cotrans-fected with R3914 viral DNA, and the tk-positive virus R7710 was isolated from the transfection progeny.

Subse-quently,

pRB4397

wascotransfected with R7710 viral DNA, and the

Oris-negative tk-negative

virus R7711wasisolated. Forconfirmationofboth the purity and the presenceof the appropriate insertions and deletions in R7710 and R7711, their DNAsand thatof R3419weredigestedwith restriction enzymes,electrophoreticallyseparated,blotted,andprobed with radiolabeled DNAs containing either sequences from the BamHI Nfragmentor aportion of thetkgene (Fig.2).

The BamHINfragmentofHSV-1(F)and R3914is -4.9 kb in length. Insertion of the tk gene and a27 promoter se-quencesintotheBamHI Nfragment expanded thefragment and resulted in a newBamHI cleavage site(Fig. 5,line 4),

dividing the BamHI N fragment into two fragments. For R7710,thesefragmentswereexpectedtobe 4.4 and 3.0 kb in size. Digestion of R3914 and R7710 DNAs with BamHI released fragments that wereofthe expected sizes and that hybridized to probe sequencesderived fromBamHIN (Fig.

2A and B, lanes 1 and 2). Furthermore, as expected, the 3.0-kb fragment from R7710 also hybridized to a probe

containingtkgene sequences(Fig. 2A, lane 3).InR7711, the smaller fragment hybridizing to the BamHI N probe was reduced to 1.3kb in size(Fig. 2B, lane 3),consistent with the deletion of the

Oris,

tk, and a22sequences.

For ensuring that the region containing

Oris

had been deleted in R7711, electrophoretically separatedBamHI and BssHII digests of R3914, R7710, and R7711 DNAs were probed with sequences from the BamHI N fragment (Fig.

2D). As expected, the 1.7-kb BssHII fragment of

R7710,

which contained the

Oris

sequence, a22

promoter-regula-tory sequences, and part ofthe tk gene, was absent from R7711.

Absence

of Oris sequences and function in R7711. The minimum sequencerequired forthe

Oris

replication function is quite small (6, 36); the smallest HSV-1 DNA

fragment

shown to possess replication origin function is 67 bp in length (6). Such a small sequence, ifretained in R7711

by

some rearrangement, mightnot bedetectedbythe

relatively

low resolutionanalysesdescribed above. For

addressing

this issue, the same DNAblot as thatshownin lanes 1to3 in

Fig.

2B was probed with anoligodeoxynucleotide probe contain-ing

Oris

sequence (Fig. 2B,lanes 4 to6). The sequence of the 50-mer oligodeoxynucleotide probe (Fig. 3, line 2) is con-tained within theminimalHSV-1 DNA segmentrequired for

amplification by viralfactors, andmutationsinthissequence abolish the ability of the DNA to act as anorigin (18,

40).

The BamHI N fragment (4.9 kb) ofR3914 DNA

(Fig.

2B,

lane 4) and the 3.0-kb fragment derived from the chimeric BamHI N fragmentofR7710 DNA (Fig. 2B, lane5)

hybrid-ized with this probe,consistent with thelocalization of

Oris

on these fragments. Theabsence ofotherhybridizing bands

is consistent with theabsence of

Oris

attheother endof

Us.

In contrast, R7711 DNA failed to hybridize with this probe

(Fig. 2B, lane 6), suggesting a complete loss of

Oris

se-quences from this virus.

An additional hybridization experiment was carried out with a shorter oligodeoxynucleotide probe to detect simul-taneously both

Oris

and OriL sequences (Fig. 2C). The 32-mer oligodeoxynucleotide (Fig. 3, line 3) is shorter in length and shows greatersimilarity to

Ori,

than thatshown in Fig. 3, line 2, and used in the hybridization experiments described above. InBamHI digests ofHSV-1(F) DNA, this

probe detected the

Oris

sequences as two stronger bands (BamHI N and Z fragments) and the OriL sequence as a weaker band (BamHI V fragment). Both R3914 and R7710 yielded only one stronger band in addition to the weaker band, indicating that theseviruses possessed only one copy J. VIROL.

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ooc co C)

1 2 3 4 5

FIG. 6. Transient replicationassay. Autoradiographic image of labeled pGEM-3Zf(+) DNA hybridizedtoelectrophoretically

sepa-rated BamHI andDpnI digestsof DNA extractedfrom Vero cells transfected with test plasmids and infected with HSV-1(F). Test plasmidswere pGEM-3Zf(+) (lane 2), pRB175 (lane 3), pRB4488 (lane 4), and pRB4489 (lane 5). Lane 1, linearized pGEM-3Zf(+) size standard. Thepositionsof markerDNAs,thesame asthose shown inFig. 2, areshowntotheleft.

of

Oris

in addition to OriL. R7711 yielded only the band corresponding to the BamHI V fragment, indicating that R7711 did notretain any

Oris

sequence. Differences in the intensity of the BamHI V band among viruses reflected variations in theamountof viral DNA loadedon each lane.

Totestdirectlyforaloss of

Oris

function,wetested DNA fragments spanning the mutagenized regions in a transient

replicationassay. The BamHI DNAfragments spanningthe mutagenized regionsfrom the left andright (withrespect to theprototypegenomearrangement)ends of

Us

(Fig. 5)were

cloned from R7711 DNA intovectorpGEM-3Zf(+)toyield pRB4488 and pRB4489, respectively. Restriction enzyme

mapping of these plasmids further verified the absence of

both

Oris

sequences (data not shown). Vero cells were

transfected with plasmids and subsequently infected with

HSV-1(F). At 13 h postinfection, total cellular DNA was isolated, digested with BamHI and DpnI, electrophoretically separated on an agarose gel, and probed with radiolabeled pGEM-3Zf(+) DNA (Fig. 6). BamHI cleaves each plasmid DNA to release a vector-length fragment, regardless of whether the DNA has replicated, whereas DpnI can cut at multiple sites only in unreplicated DNA. Thus, replicated DNA is detected as a DpnI-resistant, vector-length linear fragment, and unreplicated DNA is detected as small frag-ments. Thepositive control plasmid, pRB175, whichcarries theHSV-1(F) BamHI Z fragment containing one copy of the

Oris

sequence,yielded DpnI-resistant DNA (Fig. 6, lane 3), indicating that it contained an active origin. In contrast, neither of theplasmids carrying the mutagenized fragments cloned from R7711 yielded DpnI-resistant DNA (Fig. 6, lanes 4 and 5), demonstrating that they did not contain sequenceswithdetectable origin activity.

The results of these studies indicated that R7711 DNA lacks both

Oris

sequences and function. The results also demonstrated that the SV40 DNA replication origin con-tained in the SV40 promoter-a4 fusion could not support transient plasmid replication during HSV-1 infection. This result wasconsistent with the observations of Heilbronn and ZurHausen(12),whoshowed that stimulation of theactivity ofthe SV40replication origin byinfection with HSV-1 was

absolutely dependentonthe presence of Tantigen supplied

in trans.

Time courseofDNAsynthesis.Thesuccessful isolation of aviruslacking both copies of

Oris

demonstrates that

Oris

is not essential for viralreplication, but it seemed likely that the loss of these sequencesmightalter eithertheamountof

viral DNA synthesized, the time course of synthesis, or the

timingofreplicationof differentregions of the genome. For

testing these possibilities, Vero cells were infected with 5 PFU of viruses HSV-1(F)A305, R3914, and R7711 per cell, and total cellular DNAwasharvested fromduplicate dishes at 3, 6, 9, 12, and 15 h after infection. The DNAs were digested withBamHI, electrophoretically separated, trans-ferred to a nylon membrane, and hybridized with excess labeledprobe(Fig.7and8).The accumulation of viral DNA was assessed with two probe sequences: (i) the BamHI T fragment, located in the L component (0.273 to0.292 map

units) and distant from both OriL and

Ons,

and (ii) the BamHI X fragment, located in the S component (0.938 to 0.951 mapunits) andnear oneof the

Ons

sequences.

TheamountsofDNAdetectedfor all virusesat3 hafter infection inthisexperimentcouldnotbedifferentiated from those observed at 3 h after infection in the presence of

ime after infection 3 6 19 12 15

Copis

o O CopiesofOrison infectingvirus 2 BamHITProbe

1 0 2 1 0 2

Bam Hi X Probe

[image:7.612.150.218.76.309.2]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

FIG. 7. Autoradiographic imagesof the timecourseof viral DNAsynthesis.Shown is arepresentative autoradiographicimageof BamHI digestsof DNAs extractedfrom Vero cells infected withHSV-1(F)A305 (lanes1, 4, 7, 10,and13),R3914(lanes2, 5, 8, 11,and14),and R7711 (lanes 3, 6, 9, 12,and15)andprobed successivelywithexcesslabeledplasmids containingtheBamHIT(pRB179[1])or X(pRB124 [16]) fragment ofHSV-1(F). The time after infection atwhich each sample was harvested is indicated above the lanes. Excess probe was demonstratedby hybridization to serial dilutionsof unlabeledprobeonthesameblot(datanotshown).

0

2 2 1 0

dbu&bl

1 0

1

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2130 IGARASHI ET AL.

2 4001 1/200 /

2 ~~~~~~~~~~~~~0)

200- 100

0-- 0

0 3 6 9 12 15 0 3 6 9 12 15

Timeafter infection (hours)

FIG. 8. Graphicrepresentation of the timecourseof viralDNAsynthesis. (A)Theamountof labeled BamHI Tfragment hybridized (in countsperminute)to HSV-1(F)A&305,R3914, andR7711 DNAsrecovered from infected Vero cellsas afunction of timepostinfection (in hours)isshown. Each timepoint is themeanoftwoindependentinfections. (B)Asinpanel A,except that the BamHI Xfragmentwasused asthehybridizationprobe.

cycloheximide (datanotshown). Therefore, theamountsof DNA detected at 3 h indicated the levels of unreplicated

input DNA. Amplificationof DNAcould be detected in all infected-cellsamples harvestedat6 hpostinfection,

suggest-ing that the deletion ofone orboth copiesof

Oris

didnot cause adelayin theonsetofviral DNAsynthesis. Although

theamountsof viral DNA madebyR7711werereducedat9

h, all three viruses showed similar time courses of DNA

accumulation andproduced nearly equal amounts of prog-eny DNA. These results indicate that the viruses lacking

both

Oris

sequences are not significantly or demonstrably impairedwith respecttotheircapacityto make viral DNA.

Effect of the deletion of both

Oris

sequences on virus

multiplication. Vero cells grown in 25-cm2 dishes were infectedateitheralow(0.01 PFU per cell) or a high (5PFU

percell) multiplicityof infection withHSV-1(F)A305,R3914, orR7711. At 24 h afterinfection,cellswerelysed byasingle cycleoffreezingandthawingandsonicated,and the titer of

infectiousvirus in each lysate was determined by a plaque assay with Vero cells (Table 1). Both R3914 and R7711 showed small reductions in virus yield at 24 h. These

TABLE 1. Virus yields from Vero cells at 24 h postinfection

Virus yield,PFU/mlof culture lysate,at a Virus multiplicity of infectionofa:

5PFU/celi 0.01PFU/cell

HSV-1(F)A305

4.7x 107(1) 9.3 x 106(1)

R3914 3.3 x 107(0.70) 5.4 x106(0.58)

R7711 2.5 x 107(0.53) 2.7 x 106(0.29)

aMeanof duplicate infections. Titrationwas done withVero

cells.

The yields ofthe recombinant viruses relative to those of the parent virus, HSV-1(F)A305,areshowninparentheses.

reductions were more apparent at a low multiplicity of infection butwere at mostfourfold for R7711. Bothmutant viruses also multiplied efficiently in 143TK-, BHK, and HEp-2 cells(datanotshown).

DISCUSSION

We reportthatwehavedeleted both

Oris

sequencesfrom

HSV-1 recombinantvirus R7711. Evidence

supporting

this conclusion is based on both hybridization analyses of the viral DNAwith

specific

probesand functional assays

show-ing that the DNA sequences that in the wild-type virus

contain the

Oris

sequences are not amplified in trans by

HSV-1proteins expressedin the transfected cellsby

super-infectingvirus.

The

Oris-negative

virus R7711 yielded approximately

fourfold less infectious progeny than the parent virus from which itwasderived insingle-step growthexperimentsat a low multiplicity of infection. Cells infected with the virus

lacking both

Oris

sequences showed a demonstrable in-creasein DNAcontent at6 hpostinfection and, although the

amounts of viral DNA were clearly and reproducibly re-ducedat9hpostinfection, appearedtoaccumulate DNAat least as rapidly as cells infected with the wild-type virus thereafter. The basis of the reduced accumulation at 9 h

postinfection requiresfurtherinvestigation,but we speculate that the R7711 virus is delayed in the transition from an

early, slow phaseofreplication to a late, rapid phase. The total amounts of viral DNA accumulated in cells infected with wild-type and mutant viruses by 15 h after infection were approximately similar, suggesting that the decreased virus yield of R7711 after single-step replication did not resultdirectlyfromadefect in DNA synthesis. Indeed, it is

possiblethat the smalldifferencebetween R7711 and

HSV-1(F)A305

in virus yield is unrelated to the

Oris

deletion, J. VIROL.

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sinceourmanipulations affected thestructureofatleastfour viral genes in additionto Oris.

We conclude on the basis of our data aswell as on the

basis ofthereportspublishedpreviously (16, 25)the follow-ing. (i) Both

Oris

sequences are dispensable forviral repli-cationincells in culture. (ii)The evidence indicates that ifan

originof viral DNA synthesis is

required

forthe

amplifica-tion of a wild-type genome, 0riL is sufficient for this pur-pose. (iii) Inasmuch as the OriL sequencewas shown tobe

dispensable underconditions inwhich both

Oris

sequences wereintact (25) and inthis studyweshowed that both

Oris

sequences are dispensable, we may conclude that none of the three originsisuniquely requiredfor viralreplicationin

cellculture.Rather,ifanoriginof DNAsynthesisis

required

at all, anyone ofthe three

origins

will mostlikely suffice.

Inview of theevidencethat the absence oftwo

origins

of DNA synthesis does not

grossly

affect the

quantity

of viral DNAsynthesized, the reasonfor the evolution and

conser-vation of three origins of DNAsynthesis remains obscure. Threebroadhypothesescanaddress this

point:

(i)

Oris

and OriL mediate different types of

replication

initiation

(i.e.,

mediate the assembly of different typesof

replication

com-plexes, perhaps leading to different mechanisms of

replica-tion);(ii)thetwo

origin

typesareuseddifferentlyindifferent cells or at

specific

stages ofthe viral life cycle (e.g.,

lytic

versus latent infection); and

(iii)

these

origins

are indeed functionallyredundant,but the presence of

multiple

origins

inthe virus genome increases the

probability and/or

therate ofsuccessful initiationof DNA

replication

ordecreases the

time

required

toreplicategenome DNA.Interestingly,itcan beargued thattheplacementof eachofthe three

origins

in the HSV-1 genome could

conceivably

provide

for their

conservation

independently

of selective pressure derived

fromtheir function in DNAreplication. OriLmapsbetween

thegenesencodingthe

single-stranded

DNA

binding

protein

ICP8 and the viral DNA

polymerase.

Althoughthe

viability

of the

Ori.-negative

virus

(25)

demonstrates that the

OriL

sequenceis not essentialfor the

expression

of these genes,

the OriL sequence may contribute to proper

temporal

and

quantitative

expression.

The

Oris

sequences are present in

reiterated sequences, and their

stability

could

conceivably

bemaintainedbyasequenceconversion

mechanism,

evenin the absence ofselectivepressure.

ACKNOWLEDGMENTS

This studywas aided by Public Health Service grants from the National Cancer Institute (CA47451) and the National Institute of Allergy and Infectious Diseases (AI124009)andbyanunrestricted grant from the Bristol-Myers Squibb Program in Infectious Dis-eases.

REFERENCES 1. Baines,J.D.1992. Unpublisheddata.

2. Baines,J. D., and B. Roizman.1991. The openreadingframes UL3, UL4,UL10, andUL16 aredispensable forthegrowthof herpessimplexvirus 1 incell culture. J. Virol. 65:938-944. 3. Becker, Y., Y. Asher, E. Weinberg-Zahlering, S. Rabkin, A.

Friedmann, and E. Kessler. 1978. Defective herpes simplex

virus DNA: circular and circular-linear molecules resembling

rolling circles. J.Gen. Virol. 40:319-335.

4. Carmichael, E. P., M. J. Kosovsky, and S. K. Weller. 1988. Isolation andcharacterization ofherpes simplexvirus type 1 host rangemutantsdefectivein viral DNAsynthesis.J. Virol. 62:91-99.

5. Challberg,M. D.1986. Amethodforidentifyingtheviralgenes required for herpesvirus DNAreplication. Proc. Natl. Acad. Sci.USA83:9094-9098.

6. Deb, S., and M. Doelberg. 1988. A67-base-pair segmentfrom theOrisregionofherpessimplexvirustype 1 encodesorigin function.J. Virol. 62:2279-2286.

7. Delius, H.,andJ.B.Clements. 1976. Apartialdenaturationmap ofherpessimplexvirustype 1DNA:evidenceforinversionsof theuniqueDNAregions.J. Gen.Virol. 33:125-133.

8. Ejercito,P.M.,E. D. Kieff,and B. Roizman. 1968. Character-izationofherpessimplexvirusstrainsdifferingintheir effects

onsocial behavior of infected cells.J. Gen. Virol. 2:357-364. 9. Elias, P., C. M. Gustafsson, and 0. Hammarsten. 1990. The

origin bindingproteinofherpes simplexvirus 1 binds

coopera-tively to the viral origin of replication Oris. J. Biol. Chem. 265:17167-17173.

10. Gorman, C. M., L. A. Moffat, and B. H. Howard. 1982. Recombinantgenomes which expresschloramphenicol acetyl-transferaseinmammaliancells.Mol. Cell. Biol. 2:1044-1051. 11. Hayward,G.S.,R.J.Jacob,S. C.Wadsworth,and B. Roizman.

1975.Anatomy ofherpessimplexvirus DNA:evidence for four populationsof molecules that differ in the relative orientations of their longand short segments. Proc. Natl. Acad. Sci. USA 72:4243-4247.

12. Heilbronn, R., and H. ZurHausen. 1989. A subset of herpes

simplex virus replication genes induces DNA amplification

withinthehost cellgenome.J. Virol.63:3683-3692.

13. Jacob,R.J.,and B. Roizman.1979.Anatomyofherpessimplex

virus DNA. XII. Accumulation of head-to-tail concatamersin nuclei of infected cells and their role in thegenerationoffour isomeric

arrangements

ofviral DNA. J. Virol.29:448-457. 14. Jongeneel, C. V., andS. L. Bachenheimer. 1981. Structure of

replicating herpes simplexvirusDNA.J.Virol.39:656-660. 15. Lockshon, D., andD. A. Galloway. 1986. Cloning and

charac-terization ofOriL2, alargepalindromicDNAreplication origin

ofherpessimplexvirus type 2. J.Virol. 58:513-521.

16. Longnecker, R.,and B. Roizman.1986.Generation ofan invert-ing herpes simplexvirus1 mutant lackingthe L-Sjunctiona sequences, an origin of DNA synthesis, and several genes includingthosespecifying glycoproteinEand thea47 gene. J. Virol. 58:583-591.

17. Longnecker, R., and B. Roizman. 1987. Clustering of genes

dispensable for growth in culture in the S component of the HSV-1 genome.Science 236:573-576.

18. Martin, D. W., S. P. Deb, J. S. Klauer, and S. Deb. 1991. Analysis of the herpes simplex virus type 1 Oris sequence: mappingoffunctional domains. J. Virol.65:4359-4369. 19. Mavromara-Nazos,P., M.Ackermann, and B. Roizman.1986.

Construction andpropertiesofaviableherpessimplexvirus 1 recombinantlackingcodingsequencesof thea47gene. J.Virol. 60:807-812.

20. Michael,N.1992.Unpublisheddata.

21. Mocarski,E. S., andB. Roizman.1981. Sitespecificinversion sequenceofherpes simplexvirus genome: domainand

struc-tural features.Proc. Natl. Acad. Sci. USA 78:7047-7051. 22.

Mocarski,

E.S.,and B.Roizman. 1982.

Herpesvirus-dependent

amplificationandinversionofacell-associated viral

thymidine

kinase geneflankedbyviralasequenceandlinkedtoan

origin

of viral DNAreplication. Proc.Natl.Acad. Sci.USA

79:5626-5630.

23. Olivo,P. D., N.J. Nelson,and M. D.Challberg. 1988.Herpes simplexvirus DNAreplication:theUL9geneencodesan

origin

bindingprotein.Proc.Natl.Acad. Sci. USA85:5414-5418.

24. Poffenberger, K. L., and B. Roizman. 1985. A

noninverting

genomeofaviableherpessimplexvirus 1: presenceof head-to-taillinkages in packagedgenomesandrequirements for circu-larizationafter infection. J. Virol.53:587-595.

25. Polvino-Bodnar, M., P. K. Orberg, and P. A. Schaffer. 1987. Herpes simplex virus type 1 oriL is not

required

for virus replication or forthe establishment and reactivation of latent infectionin mice. J. Virol.61:3528-3535.

26. Post,L.E.,S.Mackem,and B. Roizman. 1981.

Regulation

of a genes of herpes

simplex

virus:

expression

ofchimeric genes

producedbyfusion ofthymidinekinase with a genepromoters.

Cell 25:227-232.

27. Post,L.E.,and B. Roizman. 1981. A

generalized technique

for

on November 9, 2019 by guest

http://jvi.asm.org/

(10)

2132 IGARASHI ET AL.

deletion ofspecific genes in large genomes: a gene 22 of herpes simplex virus 1 is notessential forgrowth.Cell 25:227-232. 28. Roizman, B., and F. J. Jenkins. 1985. Genetic engineering of

novel genomes of large DNA viruses. Science 229:1208-1214. 29. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular

cloning: alaboratory manual. Cold Spring Harbor Laboratory, ColdSpringHarbor, N.Y.

30. Sears, A. E., I. W. Halliburton, B. Meignier, S. Silver, and B. Roizman. 1985. Herpes simplexvirus 1 mutant deleted in the a22 gene:growthand geneexpressioninpermissiveand restric-tive cells andestablishment of latency in mice. J. Virol. 55:338-346.

31. Sheldrick, P., and N.Berthelot. 1975. Inverted repetitions in the chromosome of herpes simplex virus. Cold Spring Harbor Symp. Quant.Biol. 39:667-678.

32. Smith, C. A., M. E.Marchetti, P. Edmonson, and P. A. Schaffer. 1989. Herpes simplexvirus type 2 mutants with deletions in the intergenic region between ICP4 and ICP22/47: identification of nonessential cis-acting elements in the context of the viral genome. J. Virol. 63:2036-2047.

33. Spaete,R.R., and N. Frenkel. 1982. The herpessimplexvirus amplicon: a neweucaryotic defective-virus cloning-amplifying vector.Cell 30:295-304.

34. Spaete, R. R., and N. Frenkel. 1985.Theherpessimplexvirus amplicon: analysis of cis-acting replication functions. Proc.

Natl.Acad. Sci. USA 82:694-698.

35. Stow, N. D. 1982.Localizationofanorigin ofDNAreplication within the TRs/IRs repeatedregionof the herpessimplex virus type 1genome. EMBO J. 1:863-867.

36. Stow, N. D., and E. C. McMonagle. 1983. Characterizationof theTRs/IRsorigin of DNAreplication of herpes simplexvirus type 1.Virology 130:427-438.

37. Vlazny, D. A., and N. Frenkel. 1981. Replication of herpes simplex virus DNA: location ofreplication recognition signals within defective virus genomes. Proc. Natl. Acad. Sci. USA 78:742-746.

38. Wadsworth, S., R. J. Jacob, and B. Roizman. 1975. Anatomyof herpessimplexvirus DNA.II. Size,composition, and arrange-mentof inverted terminal repetitions. J. Virol. 15:1487-1497. 39. Weir, H. M., J. M.Calder,and N. D.Stow. 1989.Binding of the

herpessimplex virustype 1 UL9gene productto an origin of viral DNAreplication.Nucleic Acids Res.17:1409-1425. 40. Weir, H. M., and N. D. Stow. 1990. Twobinding sites forthe

herpessimplexvirustype1UL9proteinarerequired forefficient activity of theOris replicationorigin. J. Gen. Virol. 71:1379-1385.

41. Weller, S. K., A.Spadoro, J. E. Schaffer, A. W. Murray, A. M. Maxam, and P. A. Schaffer. 1985. Cloning, sequencing, and functionalanalysis of OriL,aherpessimplexvirus type 1origin of DNAsynthesis. Mol. Cell.Biol.5:930-942.

J. VIROL.

on November 9, 2019 by guest

http://jvi.asm.org/

Figure

FIG.6withinbetweenpRB3094parentheses)thebeTranscribedandinresultingenhancer-promoter2,HSV-13,deletionsrecombinantdepictedofcontainedpRB3906,recombinationusedSV40theRestriction expanded this diagram Oris 1
FIG. 2.BamHIwithbandfirstcontainsfragmentcontainseachthecleotide(C) Autoradiographic images of electrophoretically separated digests of wild-type and recombinant viruses probed for structure
FIG. Bamlitimages Z of 4. Sall Autoradiographic digests (lanes 1 images to 3), gene BamHI analyses the digests of the genome
FIG. 5.junctionviruswhichBstEII;yieldbetweenindicatedfragmentsLinearrangement.Thecatedrectangles,thethatR7711codingLinethesequencefragmentment Summary of genome structures of recombinant viruses
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References

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