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Construction and properties of a recombinant herpes simplex virus 1 lacking both S-component origins of DNA synthesis.


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Copyright © 1993, American SocietyforMicrobiology

Construction and




Recombinant Herpes Simplex

Virus 1




Origins of



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


mapsin the Lcomponent,whereastwosequences



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



have beendeleted, andshowthat the


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


inR7711, the a47 geneand the 5' untranscribed and transcribed

noncoding regions of the


gene weredeleted,oneof the a4promoter-regulatoryregionswasreplaced with

thesimianvirus40promoter,and the at22promoterwassubstituted withtheav27promoter.The totalamount

ofviral DNAsynthesizedinVero cells infected with the

Oris-negative (Oris-)


seenincells infectedwith the


virus. However, cells infectedwith the


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


progeny virus wasreduced at most fourfold.Althoughasingle


(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


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



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


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


sequences map

within therepeatsflanking the uniquesequence


ofthe S

component. BothOriLand


are locatedbetween diver-gentlytranscribedgenes; OriLislocated betweenthe UL29

and UL30 genes, whereas


maps between the a4gene

andeither the a22orthea47gene. Thetwo


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

(15, 41).The evidence that both


sequencesand the


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


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



Inpreviousstudies,workers inourlaboratory and

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



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


sequencesfromthe HSV-2genome were

unsuccessful,andit has beensuggestedthatatleastone


sequenceis essential for viral DNAreplication (32).In this

paper, we reportthe properties ofagenetically engineered

virusfromwhich both


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


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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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





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



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


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


sequences are deleted, pRB4325 was digested with

BssHII, andthe larger fragmentwasrecircularized with T4

DNA ligase.Two DNA fragments withdeletionsacross


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


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


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


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


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


triphosphates by use of a nick

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


probe was labeled at its 5' terminus with


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


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).


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


sequence,wesystematically deleted both


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



---_--- -' _


r rs




0 _ i 4 a47 (


kI ( 2

H NEBaS Ba Na E S BaBst N S



VIRUS 2125 B.Viruses







TK -o [

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

S Ba Bst P/NE Ba S Ba


Nd P


a47 *40

a4 Us51 _- I


Us1o( _I

II A Ii 3

H/S Ba NaE S BaBst E (E)

d a bc

a -'L- - d _ __

-a d 'c '



IS a S A

S Ba Na E (E)














I S Ba/P H/S

- -Bst/Nd

- a4


Ba Na

- a4

Ba Na


Ba Sp P H/S Ba




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


sequences within the

invertedrepeats ofthe Scomponent,i.e.,between



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



Construction of HSV-1 lacking one copyof


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


sequencewas isolated. Theparentvirus used for this


2 Na




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N cr

l- 11




cn -gr cc =



C) r-- N lce CN

_4_ -* b

- -4




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


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


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


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


andshown inFig. 3.

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

digestion. If the recombinationyielded theconstructshown





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.




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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


f- -,



'S , uS

~u :

1 2 3


4 5 6


7 8 9 10 11 12


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


sequencediscussed ingreaterdetailbelow,the

a47gene, and the5' untranscribed and transcribed

noncod-ingsequencesof the


genewere deleted.

Theviability of R3914 was consistent with theprevious observations thatonecopyof


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


-- E



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Jl UL us

,_____ ______---@, _

Oris _- IO°fiO

a4 V - a22 a0-4u7a4

Ba S Ba Bss N Ba Ba BaBstNBss Ba S Ba


USIO a a4

Ba S BaBss N Ba Ba Psv4O Ba


Oris TK

a4 _1W- 0HOW' _ a22

ab a

Ba SBaBss Baa Ba Ba


a4 in-4 I-*. b a

Ba SBaBssBa


L 11.J



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


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


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.



is located in the promoter-regulatory

sequencesof both the


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

In making insertions and deletions around the remaining

copy of


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


gene were insertednear


in thea22genein the BamHI

N fragment such that thetk gene wasunder thecontrol of the


promoter, whereas the a22 gene wasunder the control

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




promoter-regulatorysequences, and the


selectable markerweredeleted. The deletion in pRB4397

didnotremove anyTAATGARATmotifs, important fora4

gene expression, from the region upstream of the a4 gene

promoter. The


insertion plasmid pRB4325 was

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



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


tk, and a22sequences.

For ensuring that the region containing


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


which contained the


sequence, a22

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


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


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


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


some rearrangement, mightnot bedetectedbythe


low resolutionanalysesdescribed above. For


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


2B was probed with anoligodeoxynucleotide probe contain-ing


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,


The BamHI N fragment (4.9 kb) ofR3914 DNA



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


on these fragments. Theabsence ofotherhybridizing bands

is consistent with theabsence of


attheother endof


In contrast, R7711 DNA failed to hybridize with this probe

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


se-quences from this virus.

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


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


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


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.

on November 9, 2019 by guest



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.



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


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

Totestdirectlyforaloss of


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


(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



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


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


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


demonstrates that


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


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



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


o O CopiesofOrison infectingvirus 2 BamHITProbe

1 0 2 1 0 2

Bam Hi X Probe


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).


2 2 1 0


1 0


on November 9, 2019 by guest




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


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



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

Effect of the deletion of both


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


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


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).


We reportthatwehavedeleted both



HSV-1 recombinantvirus R7711. Evidence


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


probesand functional assays

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

contain the


sequences are not amplified in trans by

HSV-1proteins expressedin the transfected cellsby




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


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


in virus yield is unrelated to the


deletion, J. VIROL.

on November 9, 2019 by guest


[image:8.612.112.500.76.319.2] [image:8.612.57.299.630.701.2]

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


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

originof viral DNA synthesis is



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


sequences wereintact (25) and inthis studyweshowed that both


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

cellculture.Rather,ifanoriginof DNAsynthesisis


at all, anyone ofthe three


will mostlikely suffice.

Inview of theevidencethat the absence oftwo


of DNA synthesis does not


affect the


of viral DNAsynthesized, the reasonfor the evolution and

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




and OriL mediate different types of




mediate the assembly of different typesof


com-plexes, perhaps leading to different mechanisms of



typesareuseddifferentlyindifferent cells or at


stages ofthe viral life cycle (e.g.,


versus latent infection); and




are indeed functionallyredundant,but the presence of



inthe virus genome increases the

probability and/or

therate ofsuccessful initiationof DNA


ordecreases the



toreplicategenome DNA.Interestingly,itcan beargued thattheplacementof eachofthe three


in the HSV-1 genome could



for their



of selective pressure derived

fromtheir function in DNAreplication. OriLmapsbetween






ICP8 and the viral DNA




of the




demonstrates that the


sequenceis not essentialfor the


of these genes,

the OriL sequence may contribute to proper







sequences are present in

reiterated sequences, and their






evenin the absence ofselectivepressure.


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.

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on November 9, 2019 by guest



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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