Novel rearrangements of herpes simplex virus DNA sequences resulting from duplication of a sequence within the unique region of the L component.

Vol.53,No. 2 JOURNALOFVIROLOGY, Feb. 1985, p.456-461

0022-538X/85/020456-06$02.00/0

CopyrightC 1985,American Society for Microbiology

Novel

Rearrangements

of

Herpes

Simplex

Virus DNA Sequences

Resulting from Duplication of

a

Sequence Within

the

Unique Region

of the

L

Component

KAY L. POGUE-GEILE, GLORIAT.-Y. LEE, ANDPATRICIA G. SPEAR* Department ofMicrobiology, University of Chicago, Chicago, llinois 60637

Received 29May 1984/Accepted 15 October 1984

We constructed insertion mutants of herpes simplex virus type 1 that contained a duplication of DNA sequences from the BamHI-L fragment (map units 0.706 to 0.744), which is located in the unique region of the Lcomponent (UL)ofthe herpes simplex virus

type

1genome. The second copy of the BamHI-L sequence was inserted ininverted orientation into the viral thymidine kinase gene (map units 0.30 to 0.32), also located Within UL. Asignificant fraction of the progeny produced by these insertion mutants had genomes with rearranged DNA sequences, presumably resulting from intramolecular or intermolecular recombination between the BamHI-Lsequences at the twodifferentgenomic locations. The rearranged genomes either had an inversion of theDNA sequenceflankedby theduplication or were recombinant molecules in whichdifferentregions ofthe genome had been

dfplicated

and deleted. Genomicrearrangements similar to those described here have been reported previously but only for herpes simplex virus insertion mutants containing an extra copy of the repetitive a sequence. Such rearrangements have not been reported for insertion mutants that contain duplications of herpes simplex virus DNA sequences from largely unique regions of the genome. The implications of these results are discussed.

The genome of herpes simplex virus (HSV) is 96 x 106

daltons (Da) and is composed oftwo segments, long (UL) and short (Us), of unique DNA. These two segments are each flanked by inverted repeats, the ends of which are common toboth segments andarerepeated in direct

orien-tationatthe endsof thegenome (4, 18, 20, 26, 28)(seeFig.

1).Recombination apparentlyoccursbetweentheseinverted

repeats, resulting in the inversion ofthe unique segments

flanked by the repeats. Because UL and

Us

can invert

independently ofeach other, populations ofHSV genomes usually contain equimolar quantities of the four possible isomers (5, 10, 29, 37).

Previous studies showed that, when specific HSV type 1

(HSV-1) DNA sequences are duplicated by insertion of a

second copyintothethymidine kinase(tk) gene of the virus,

inversions ofthe DNA sequence flanked by the duplicated

sequence can occur. These inversions could be detected in the genomes ofprogeny virus, provided that the inserted

sequence and the sequence at the normal locus were in

inverted orientation and that the inserted sequence

con-tained the repetitive sequencedesignated a(17, 18, 20, 30).

Inversions could not be detected when inserted sequences were from unique regions of the genome (7, 14, 17). We report here, however, that duplication ofa sequencefrom

the UL component can also result, at apparently high fre-quency, either in inversionofthe DNAsequences flankedby

invertedcopiesoftheduplicationor in other DNA rearrange-mentsresultingfromintermolecular recombinationbetween an inserted BamHI-L sequence and the same sequence at thenormallocus.

Thestudies described here wereinitiated for the purpose

ofcharacterizing a putative

fISV-1

gene product that gov-erns theprocess ofHSV-inducedcellfusion. Mutant

HSV-* Correspondingauthor.

tPresent address: School of Medicine, University of Miami, Miami, FL 33101.

1(MP) causes the fusion of infected cells under conditions in which awild-type sibling strain,HSV-1(mP), does not (12). The mutation was mapped first to mapunits 0.70 to 0.83(27)

and more recently to map upits 0.735 to 0.740 (2, 22), a region contained within the BamHI-Lfragment of the HSV-1 genome. Insertion mutants were constructed which con-tained the

BamHIH-L

fragment ofHSV-l(mP)DNA inserted into the tk gene ofHSV-1(MP) DNA and vice versa; this was done tocharacterize aproductexpressed from the insert that could either cause HSV-1(mP) to induce cell fusion or complement the mutation of HSV-1(MP). The insertion mutantsisolated did not serve the original purpose,because marker rescue and marker transferfromtheinsertedlocus to the normal locus(and viceversa) appeared to occur at high frequency, possibly by mechanisms proposed and discussed

previously (4, 13, 32). This precluded the

correlation

of phenotype with expression of products from the inserted DNA. This study therefore focuses on the novel DNA arrangementsoccurringin these mutants.

MATERIALS ANDMETHODS

Cells and viruses. Vero and HEp-2 cells were obtained from theAmerican Type CultureCollection, Rockville,Md.

HEp-2cells weregrown in DulbeccomodifiedEaglemedium with 10% fetal calf serum, and Vero cells were grown in medium 199with5%fetal calf serum. The viralstrainsused wereHSV-1(MP) andHSV-1(mP) (6, 11, 12).

Plasmids. All vector sequences present in the plasmids

used in

thi5

study were from pBR322 (1). pRB142 and

pRB103,containingthe BamHI-D' andBamHI-Q fragments, respectively, from HSV-1(F) DNA (23), were kindly

sup-plied by Bernard Roizman, University of Chicago. pKPG103R was subcloned from pRB103 by digestion with

SalI and BgIlI followed

by

religation

at

37°C

for 1 h. To obtain pKPG103L, pRB103 was digested with HindIII and

BglII, thelargerfragmentwas isolatedfromanagarosegel,

and the ends were filled in with DNApolymerase I Klenow fragment

Iand

religated at 16°C for 8 h(15). pKPG103R and 456

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REARRANGEMENTS OF HSV DNA SEQUENCES 457

mapunits I.. 0.0

. a b

HSV-I(MP) C2Z

, , , -I

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

tk _L - b aac' S ca

pTK 106

Ba

a l HSV- (mP) Iii

Bg/Ba Ba/Bg Ba

b tk Lt

_~~~--_ ~

_ -

:

ll

*

aa'c S ca

pTK 133

Ba Bg/Ba Ba/Bg Ba

molecular

weights - 1.7 -3.5 1 0.5H

FIG. 1. Mapsof the HSV-1(MP) andHSV-1(mP)genomesand of theplasmids used in these studies. Theorganizationof thegenomeswith respect toUL andUsand therepetitive sequences (LI )is shownfor bothHSV-1(MP) and HSV-1(mP). Thetkgene (i ),the BamHI-L sequenceofHSV-1(MP) (

M

),andtheBamHI-LsequenceofHSV-1(mP) (@ )areshown in theappropriate viralgenomesandplasmids. The arrows indicate the directions of the two BamHI-L sequences withrespect to eachother in the insertion mutants. The restriction endonucleases used for cloning these HSV-1 sequences were BglII (Bg) andBamHI (Ba). To construct pTK133, the cloned BamHI-L sequencefromHSV-1(MP)presentinplasmid pTYL133wasrecloned into thesingle BgII site of the tkgeneinpRB103. Thesameprocedure was used to construct pTK106, except that the BamHI-L fragment was from pTYL106, which contains the BamHI-L fragment from HSV-1(mP). TheBglII-BamHI (Bg/Ba) ligation joints are resistant to cleavage by bothenzymes. The sizes(106 Da) ofthe ligatedDNA fragments areindicated.

pKPG103L contain theBamHI-Q sequences thatare to the right and left, respectively, ofthe single BgIII site in this fragment (Fig. 1). pTK133andpTK106contain the BamHI-Lfragment (mapunits 0.706to0.744)fromHSV-1(MP)and HSV-l(mP) DNAs, respectively, insertedinto theBgIII site ofpRB103. Construction of these plasmids isdescribed in thelegendtoFig.1.pKPG101containsaSall fragment (map units 0.702to0.752) fromHSV-1(MP)DNA. pKPG101 was digestedwith BamHI andreligatedtoformpKPG102,which contains HSV-1 DNAsequencesfrom the BamHIsitetothe SalIsiteat0.744 and 0.752mapunits, respectively.

Construction of recombinant viruses. Rabbit skin cells werecotransfected eitherwithmixtures ofHSV-1(MP) DNA and BamHI-digested pTK106 DNA or with mixtures of HSV-1(mP)DNAandBamHI-digested pTK133DNA (8,14, 36). When thetotalcytopathiceffectwasobserved,progeny

viruswereplated underagarose containingthymidine arabi-noside (araT) at 100 ,Lg/ml (17). A second plating under

agarose with araTwasdone to purifythe virus.

DNApreparationandanalysis. Plasmid DNAwaspurified in cesium chloride gradients by equilibrium centrifugation (25) of cleared lysates (9). HSV-1(MP) and HSV-1(mP) DNAsused for transfections orcloningwere purified in Nal

density gradients as described by Walboomers and ter Scheggt (35) and modified by Zezulak and Spear (38). Recombinant viral DNAs usedfor Southern blotting exper-iments were obtained from Vero cells infected with

recom-binantvirus that had beenplaque purifiedtwice. These viral DNAs wereprepared by phenol extraction ofthe cytoplas-mic fraction of infected cells lysed with Nonidet P-40 (38). The DNAs weredigested with restriction enzymes as

spec-ified by the manufacturer (New England BioLabs, Inc.), electrophoresed on agarose gels, and blotted to nitrocellu-lose(31). Hybridizations were doneat65°Cin 6x SSC(0.9 M NaCl and0.09 M sodium citrate)-30% formamide-salmon

sperm DNA (50 p.g/ml)-lx Denhardt solution (15)-sodium dodecylsulfate 0.5% for 8to15 h. Nitrocellulosefilterswere

washed three times in 2x SSC, one time in lx SSC, and

finally in 0.4x SSC. Nick-translation kits (New England

NuclearCorp.) wereusedto make 32P-labeled DNAprobes fromundigested plasmids.

RESULTS

Selectionandcharacterizationof viruses witha duplication

of the BamHI-L sequence. pTK133 and pTK106 were

con-structedbyinsertingthe BamHI-Lfragment of either HSV-1(MP) or HSV-1(mP) DNAinto the single BgiII site of the cloned BamHI-Q fragment present in

pRB103 (Fig.

1). Insertions ofDNAatthis

BgIII

site resultinanonfunctional tk gene (14, 16, 24, 34). After cotransfection of cells with HSV-1(mP) or HSV-1(MP) DNA and one ofthe plasmids shown in Fig. 1, recombinant viruses

containing

the

appro-priate insertion in their tk genes were selected by their

resistanceto araT(17). AraT-resistant isolateswere plaque purified, and the DNA was extracted from cytoplasmic fractionsof the infected cells. Mostof theviral DNA in the

cytoplasmic fractionswas packagedin progeny virions. DNAsfrom mostofthe araT-resistantplaqueisolates we

analyzed were shown by Southern blot analysis tocontain

the BamHI-L sequence inserted into the tk gene. The genomes ofeight of the viruses analyzed (Fig. 2) were all

missing the BamHI-Q fragment (2.2 x

106

Da); they

con-tained insteada new

fragment

(5.7x 106 Da)thathybridized

with both the BamHI-Lprobe and theBamHI-Q or tk probe and was the size predicted for the new BamHI fragment generatedby theinsertion. All viruses(includingthe parent MPstrain)hadabandat3.5 x 106 Da,whichisthe BamHI-L

fragment atits normal locationon the genome.

Two novel fragments were detected in BamHI digests of DNA from the insertion mutants. The amounts of these fragments relativetothosepredicteddiffered frommutantto mutant. The novelfragments, 5.2 x

106

and 4.0 x

106

Da, hybridized to both the BamHI-Q and BamHI-L probes.

1-t-VOL. 53, 1985

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458 POGUE-GEILE ET AL.

L Probe Q Probe

2 3 6 7 8 11 2 3 4 MP 2 3 6 7 8 11 2 3 4 MP

[image:3.612.132.493.75.305.2]

MP-TK106 mP-TK133 MP-TK106 mP-TK133

FIG. 2. Analysis ofgenome structuresof araT-resistantprogenyobtainedfrom cells cotransfected withHSV-1(MP)DNAandpTK106or

withHSV-1(mP)DNAand pTK133.BamHIrestriction fragments of virion DNA fromHSV-1(MP) and from nine araT-resistant isolateswere electrophoresedon a0.7%agarosegel and transferredtonitrocellulose. Thefragments containing sequenceshomologoustothe BamHI-L sequence(Lprobe) and theBamHI-Qsequencecontaining the tkgene(Q probe)wereidentifiedbyhybridization. The sizes(106Da)of the fragments detectedareindicated.

Their sizes were as predicted for fragments from genomes

resulting fromtherecombinationaleventsdepictedinFig.3.

Genomes with an inversion ofthe DNA sequence between the duplicated BamHI-L sequences (product 5) could be

generated by intramolecular recombination or bya double-crossover intermolecular recombination event as shown.

Many of the defective products of intermolecular

recombi-nation (products 1 through 4) could theoretically be pack-aged into virions. These products were approximately ge-nomelength (since thetwocopies of theBamHI-L sequence

in the insertion mutants were approximately equidistant from the ends ofthegenome), andthey containedcleavage andpackagingsignals that have been showntobepresent in therepetitivea sequencelocated atboth ends of thegenome and at the L-Sjunction (33;N. Frenkel, L. P.Deiss,and R. R. Spaete, Abstr. Herpesvirus 1984, no.

1357,

p. 198).

To test the hypothesis that the anomalous fragments

containing

sequences that

hybridized

to boththeBamHI-L

and BamHI-Q fragments were from recombinant genomes (Fig. 3), we digested DNA from the progeny of two insertion mutants withSail. This enzymedoes notcleavewithin the

BamHI-LorBamHI-Qsequence and generatesfragmentsin

whichsequences contiguousto theanomalousBamHI

frag-ments can be identified. Figure 4 shows the two separate

regions of the genome in which BamHI-L sequences were present in opposite orientations in the insertion mutants.

Alsoshown is thechangeincontiguousDNA sequences that

would result from recombination between thetwoBamHI-L

sequences. The sizes of theSall fragmentscharacteristic of each BamHI-L locus in parental orrecombinant form and theprobestowhichtheywouldhybridize are also indicated.

All four of the SalI fragments depicted in Fig. 4 were detected (Fig. 5), providing evidence for the predicted

recombinational events.

DNAsfrom theinsertionmutants werealsodigestedwith EcoRI or HpaI. Both of these enzymes cut within the

PARENTAL MOLECULES PRODUCTS

Inverted.Repeats a b b'a'

Intermolecular Recombinations 2)a}aa

ab b'a'ac' ca single b

crossove

b'a,

| b

Intramolecular Recombination

ab

z:j

a'c' caa b X

[= ET=

~~~~~~~~~~~~~~-a b ba'a'c' ca

5)1 - - 2 EZIJZJ m

FIG. 3. Results of intermolecular andintramolecularrecombination between the two BamHI-L sequences present at differentlociin the insertionmutants. Left, Genome structures ofthe mutantsresulting from insertion of theBamHI-L sequenceinto thetkgeneinopposite orientationtotheresidentBamHI-L sequences. Symbols: ,repetitive sequences(superscript letters refertothe samestrand ofDNA withinamolecule: a, b, and c are sequences homologous to a', b', and c', respectively); EZZ,BamHI-L sequence (orientation is shown by thedirection of thecross-hatching);X,recombination sitesgenerating the products shown on the right. The molecules are drawn to scale with respect to theparticular featuresidentified, except that a portion of each genome has been omitted from most of these representations where indicatedby breaks between parallel diagonallines.

J. VIROL.

.;_,.awl* or--5.7-

W

1:

.::. "'5.21

,4.o- #aft

l*,',.-3.5

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PARENTAL FRAGMENTS

0( L 0(r) D L 8(1)

7.8 4.8 t

NOVEL FRAGMENTS

0() L Do 0(r) L B(M)

t

~~6.5 6J

FIG. 4. Restriction fragments generated by Sall cleavages of rearranged ornonrearrangedgenomesfrom the insertion mutants. Top, Parental Sall fragments that will hybridize to a BamHI-L probe ifno recombination has taken place betweenthe BamHI-L sequences atthetwodifferent loci. Bottom, Novel Sallfragments that willhybridizeto aBamHI-L probe ifrecombination has taken place between thetwoBamHI-L sequences atthedifferentloci. The verticalarrowsshow the positions of theSall recognitionsequence; numbers between the vertical arrows indicate the sizes of Sall fragments(106Da). Horizontal arrowsindicate that theBamHI-L sequences ( E ) areininverted orientation. The letters above the lines are the names of BamHI fragments from HSV-1 DNA (or portions thereof) usedasprobes in the Southern blotanalysis (Fig. 5). Symbols: M,the HSV-1 DNA sequence contained in probe Q(l) or plasmid pKPG103L; MgE, the HSV-1 DNA sequence in probeQ(r)orplasmid pKPG103R; C,theHSV-1DNA sequence of the BamHI-D' fragmentpresent in pRB142(mapunits 0.700to 0.706); _, the HSV-1DNA sequence,designated B(l), contained inpKPG102.

BamHI-Lsequence so that no restriction fragments specific to the recombinant genomes would be generated. As pre-dicted, these restriction digests showed no novel bands (data not shown).

DISCUSSION

Evidence hasbeenpresented that the normalinversionof

the UL and Us components ofwild-type HSV genomes is due to site-specific recombination between inverted copies ofthe reiterateda sequences and is mediatedby viral gene products (3, 4, 17-21, 30). The evidencefor this site

speci-ficity

was obtained by inserting fragments from either the

unique or the reiterated regions ofthe genome into the tk geneoftheHSVgenome (17). Insertions ofDNAfromthe

reiteratedregions ofthegenome resulted in novelinversions between inverted copies of the reiterated sequences. In contrast, insertions ofthree different Sacl fragments from

UL (map

units0.64to

0.65)

or

Us (map

units0.910to0.925 or0.925 to0.935) didnotresult in anynovelDNA rearrange-ments that could be detected in cytoplasmic packaged ge-nomes.

Two other HSV DNAfragments from unique regions of

the genome, in addition to the BamHI-L fragment, have been insertedintothe tk geneofHSV. Noevidence forDNA rearrangements was obtained when a

SalI-to-BamHI

frag-ment containing the genefor gC-1 (map units 0.62 to 0.64) was inserted in either orientation into the HSV-1(MP) ge-nome (14). In contrast, when the homologous HSV-2 se-quence encoding gC-2 (map units 0.62 to 0.64) was inserted into the HSV-1(MP) genome in inverted orientation to the

gC-1gene, one of the two novel restrictionfragments which

would be predicted by inversion was detected in a

BamHI

digest

ofpackaged viralDNA(K. M.Zezulak,Ph.D. thesis,

University of Chicago, Chicago, Ill., 1983). Perhaps the second novelfragmentwasnotdetected because its size was very similar to that of aBamHIfragment fromthe parental viral DNA. Thus, it is possible that inverted duplication of

the sequence encoding gC-2 as well as the BamHI-L se-quence results in DNA rearrangements.

Several factors other than the actualfrequency of recom-bination between the duplicated sequences could influence theability to detect rearranged genomes, and these factors couldhelp to explain the different consequences of inserting the BamHI-L sequence and other unique sequences. In all the experiments described here and elsewhere, the DNA that was analyzed was DNA packaged in virions. Thus, detection of recombination products can be limited by the

requirements for packaging.Theserequirementsinclude the presence of the a sequence on at least one end of the genome (33; N. Frenkeletal., Herpesvirus 1984)and anappropriate

genomesize. Although the range ofpermissible sizes is not known withcertainty,mutantscontaining deletions or

inser-tions that are greater than about 8% of the genome length have not yetbeenreported. The detectionof recombination

products may also depend in part on the ability ofvirions

containing rearrangedgenomes toreplicate. Defective rear-ranged genomes would not be amplified during successive roundsofreplication except under conditions ofhigh

multi-plicity of infection.

It seems unlikely, however, that packaging constraints

alone canexplain the different consequences of duplicating

various sequences from unique regions of the genome. Intramolecular recombination between duplicated se-quences in opposite orientations should always yield

packageable genomes. Recombinant products that might

have resulted from intermolecular recombination in this

study (i.e., products 1through4, Fig. 3) are an appropriate

L

B(l)

Q(r)

Q(O)

D

-78 40 -6.5 6.1 MD*ft -4.8

s a

X 3 2 3 2 1 3 2 1 3 2 1 3 2 1

Hind)ll

FIG. 5. Sall restriction endonuclease digestion and Southern blotanalysis of DNA from the BamHI-L insertion mutants. This Southern blot includes Sall digests of DNA from HSV-1(MP) (sample 1)andfromtheinsertionmutantsMP-TK106-7 (sample 2) and mP-TK133-2(sample 3).Samplesof theseSall-digestedDNAs wereloaded into fourorfive wells,and theappropriatelanes were hybridizedwith the labeledprobesindicated at the topofeachpanel (as described in the legend to Fig. 4). The first panel shows

HindIll-digestedXDNAhybridizedwithlabeledX DNA. Thebands detected herewereusedassize markerstodetermine the sizes(106 Da) ofthe bands in theother.panelsasshownonthefarright.

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460 POGUE-GEILE ET AL.

sizeforpackagingonly because the tk gene and the normal genomic location of the BamHI-L sequence happen to be equidistant from the ends of the genome. This is probably also true for at least two of the other insertion mutantsfor which no evidence ofgenomic rearrangement was obtained. These insertion mutants include those described by Lee et al. (14)and Mocarski et al. (17), which had sequencesfrom

map units 0.62 and 0.64 and 0.64 to 0.65, respectively,

duplicated in the tk gene.

The possible defectiveness of rearranged genomes also does not fully explain the different consequences of

dupli-cating BamHI-L sequences and other unique sequences.

Smileyet al. (30) detected packaged molecules fromwhich half ofthe genomehad been deleted due to recombination

between twocopiesof the a sequence in direct orientation in

infectionsatrelatively lowmultiplicity (1 PFU per cell). The deletedgenomes were an appropriate size to be packaged as dimers.

It seems likely, therefore, that an unusually high rate of

recombination between theduplicatedBamHI-L sequences

isatleast partlyresponsible for the resultsreported here. It

ispossible thata site-specific mechanism is responsible for

theserearrangements, because similarrearrangements have not been detected in HSV genomes with duplications of otheruniquesequences. This argument was used

previously

to propose the existence of a site-specific mechanism for recombination between a sequences (17). If both of these genome regions contain signals for site-specific recombina-tion, then presumably the signals arefordifferent systems; or, if thesame system isused, itdoes notpromote

recom-bination between thea andthe BamHI-L sequences.

Alter-natively, the recombinational events we observed may

re-quire

homology butnot a

specific

nucleotide sequence, with

the frequency of recombination between homologous se-quences depending onfactors not yetdefined.

ACKNOWLEDGMENTS

WethankB.Roizman forprovidingtwoof theplasmids,R.Aspell forgrowing and maintaining cell lines, and J. Hoshizaki fortyping themanuscript.

This workwassupportedby grantstoP.G.S. fromtheAmerican Cancer Society and the Public Health Service (CA21776 and CA19264).K.L.P.-G. isatrainee under National Research Service Award 5 T32PHS GM07281.

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