Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus type 1 Angelotti DNA.

0022-538X/81/070075-07$02.00/0

Amplification of

a

Short Nucleotide Sequence

in the

Repeat

Units of Defective

Herpes

Simplex

Virus Type

1

Angelotti

DNA

H. C.KAERNER,* A. OTT-HARTMANN, R. SCHATTEN,C. H.SCHRODER, ANDC. P. GRAY

Instituteof Virus Research, German Cancer Research Center, 6900 Heidelberg, West Germany

Received31December1980/Accepted 8 April 1981

Ithas been shown earlier thatthe reiterated regionsTRsandIRsbracketing the

Ussegment ofherpes simplex virustype 1Angelotti DNAareheterogeneous in

size by stepwise insertion of one to six copies of a 550-base-pair nucleotide

sequence. Considerably higher amplification of this sequence was observed in

defective viral DNA:up to 14copieswere detectedtobe inserted in therepeat

units ofamajor class of defective herpes simplex virus type 1Angelotti DNA,

dDNA1, which originated from noncontiguous sites locatedinUL and the inverted

repeats of the Scomponentofthe parentalgenome. Physicalmaps were

estab-lished for the cleavage sites ofKpnI,PstI,XhoI, and BamHI restriction

endonu-cleasesontherepeatsof dDNA1. Themapposition of the insertionsequence was

determined. Itwasdemonstrated that theamplified insertswere notdistributed

at random among or within the repeats. A given total population of dDNA1

molecules consistedof differenthomopolymers, eachof which containeda

con-stant number of inserts in all of its repeats. Assuming that a rolling-circle

mechanism is involved in thegeneration of full-length defective herpes simplex

virustype 1AngelottiDNAfrom singlerepeatunits,these data suggestthat the

550-base-pairsequence isamplifiedin therepeatsbefore thereplicationprocess.

Virionscontainingdefective viral DNAappear

in the virus offspring in the course of serial

passagesof herpesviruses athighmultiplicities

of infection(1-7,9,11-14, 17,18, 20). The

defec-tive DNAhas, approximately,aviralDNA unit

lengthandis madeupby repetition ofrestricted

portions of the parental genome. The present

study is concerned with one major classof

de-fective herpes simplex virus type 1 Angelotti

(HSV-1 ANG) DNA, dDNA1, which has the

samebuoyant densityasthe parental viralDNA

(17). dDNAl hasarepeat sequenceof

approxi-mately7x

10'

daltons and

originates

from

non-contiguous sites of the parentalgenomelocated

between0.33to0.42 and either0.82 to0.857 or

0.963 to 1.0 mapunitsontheprototypeisomer

(9). As has been reportedearlier, the reiterated

regionsTRsandIRsof theScomponentof

HSV-1ANG standardDNAcontainone tosixtandem

insertionsofa550-base-pairsequenceunit.

Con-sequently, theS-terminal and the L-Sjoint

re-striction fragments appear as series of DNA

bandsin agarosegels, equidistant byabout 0.35

x

10'

daltons. By investigating the sequence

arrangement ofHSV-1 ANGdDNA1,wefound

that, in some of the repeats of this class of

defective DNA, this sequence is amplified as

muchas14-fold. One questionarisingfrom this

findingwaswhether the insertsweredistributed

atrandom within andamongtherepeatunits of

individual dDNAl molecules or whether the

totalpopulation of dDNAl molecules consisted

of differenthomopolymers madeupofidentical

repeatscontainingconstantnumbers ofinserts.

To distinguish between these possibilities, we

partiallydigested dDNAl withanumber of

re-striction endonucleases and examined the

di-gestsfor theappearance andsizedistribution of

intermediatefragments. Most of theenzymes we

usedrenderedsmearswhichcouldnotbe

inter-preted. PstI, however, cleavedpreferentiallyon

one of its two cleavage sites on the dDNA1

repeats. From the fragmentpatterns ofpartial

PstI digests we concluded that dDNAl repre-sents a mixture of different homopolymers. Physical mappingof thecleavagesites of various

restriction enzymes on the repeat sequence of

dDNAl further revealed that all of the

homo-polymers have common left-hand and right-handtermini.

MATERIALS AND METHODS

Virus andcells.HSV-1 ANG(1,13,15)was

prop-agatedonAfrican greenmonkeykidneycells(RC-37

Rita,Italdiagnostics, Rome, Italy)asdescribedearlier

(17).

75

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dDNA1. The development of virus particles

con-taining dDNA1 during serial virus passages at high

multiplicities of infection and the isolation of the

ECORI-, HpaI-, and HindIII-resistant dDNA1 from

maturevirions has been described in detailpreviously

(9, 17).In afinal step, defective DNAwaspurifiedas

DNAsedimentingatthesameapparentrateinneutral

sucrosegradientsasthatof viral standard DNA.

Restriction endonucleases. BamHIwas a

prod-uctof BRL,Inc.,Rockville,Md. All other restriction

endonucleases were purchased from New England

Biolabs, Boston, Mass. Enzyme digestionswere

per-formedby theprescription of theproducers.

Gelelectrophoresis of dDNA1 restriction

frag-ments.Electrophoresiswascarriedoutin0.6 or0.8%

vertical agarose (Seakem, Richmond, Calif.)slabgels

at 40V for18hat roomtemperature. Thegelswere

then stained with ethidium bromide andphotographed

under UVlight.

RESULTS

Evidence of multiple insertions of a

550-base pair nucleotide sequence dDNAl.

dDNA1 was digested with various restriction

enzymes, and the fragments were separated in

Xho

I

Pst I

agarose gels. Completion of the digestions was

carefully tested as follows: excess enzyme was multiply addedat 2-hintervals afterthe first

2-hdigestion period, and, in parallel assays, diges-tions were continued for 24 h as controls. The cleavage patterns obtained with XhoI, PstI, BamHI, and KpnI (Fig. 1) represented complete digestion in eachcase.

Allof the patternsdisplayed remarkable "step ladders" of DNA fragment bands, starting with base fragments of different molecular weights. The individual steps differedby 0.35 x 106

dal-tonsineachcase.Thisfinding suggests that the

repeat sequences of dDNAl contain multiple insertions of 550-base-pair sequences. The dif-ferent intensities of the individual step frag-ments further suggest that the number of dDNAl molecules present decreases with the increasing number of insertions that they con-tain.

Sequence or organization of the repeat

units of HSV-1 ANG dDNAl. In addition to

the majorstepladders, all of the fragment

pat-BamH

I

-A -B

5.71

5,35

5.0 * 4,77 *4.4 *4,01

-C

-E 1-t> 1.05

0.94

5,05 -4,7 -4

357

4,35----0 4,1

t *3,75

-A -o 3'4

t -.

3,05--B -o2,7

* 2,31 --f>2,08-.

1,6

-c -D

WV WV

'WV

t~~~~~~~~~1

&:'S:m:

-A 53

4.95

-4,6

-*4,05 *3,7

-C 2,65

--lo2,3 -D

1,95

41,6 6 _

1.4 _ - D

FIG. 1. DigestionofHSV1ANGdDNAIwiththe restriction endonucleases XhoI, PstI, BamHI, and KpnI.

Ethidium bromide-stained restrictionfragments electrophoretically separatedondifferentagarosegels (0.5 to 1%). The numbers represent the molecular weights x 106 of thefragments which were estimated by

calibratingthegelsusing HindIII restriction fragments ofADNA(19) and simian virus40DNA (15)and

HaeIIIrestrictionfragments of gX174RF DNA (10)asmarkers. Openarrows,left-handterminalfragments; Filledarrows, right-handterminalfragments.

5.25-4.9 4.55-*4.2 *3,85 *3.5

Xi>1.15

0.8

0.54

-A

-B

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REPEAT UNITS OF DEFECTIVE HSV-1 ANG DNA

ternsshown inFig. 1(andanumberofpattems

resulting from other restrictionenzymes which

are notshown) containeda second minorstep

ladder offragments (XhoI-B, PstI-B, BamHIB,

andKpnI-C)whichalsoareequidistantby 0.35

x

10'

daltons.Furthermore,each of thepatterns

containedoneminorfragment (XhoI-C, PstI-C,

BamHI-C, and KpnI-B) which when added

to-gether with the base stepof the corresponding

minor step ladder, equals the molecular weight

of the base step fragment of the major step

ladder. The molarities of the DNA fragment

bands were estimated from their molecular

weights andby optical scanning of the stained

gels. By taking the molarity of the discrete minor

fragment(indicated by theopen arrowinFig. 1)

for the individual pattern as one (i.e.,onlyone

fragment ispresent per dDNAl molecule), we

found the sum of the molarities of the minor

stepladderfragmentstobeapproximatelyone.

Itwasassumed that the fragments of the minor

stepladders and the 1 M fragmentsin eachof

theindividualpatternsshown inFig.1represent

left-hand and right-hand terminalfragments of

thecomplete dDNA1 molecules. This

assump-tion implies that the left-hand terminal

frag-Left Hand Terminus

mentscreatedby XhoI, PstI, BamHI,and KpnI

do not contain multiple 550-base-pair inserts,

whereas all of thecorresponding right-hand

ter-minal fragments do, and, hence, each of the

latter is displayed as a step ladder of bands in agarosegels.

The fragments XhoI-D, XhoI-E, PstI-D,

BamHI-D, and KpnI-D, all ofwhich were more

than10 M asjudgedbyopticalscanning, were

consideredto representinternalsegments of the

dDNA1 repeat sequenceunits. The same high

molarity was determined for the sum of the

major step ladder fragments (XhoI-A, PstI-A,

BamHI-A,andKpnI-A), which apparently also

representinternal DNA fragments.

From the above data, the sequence

arrange-mentof the dDNA1 repeatsand themap

posi-tions of the KpnI, XhoI, PstI, and BamHI

cleav-agesitesonthe repeatunitswere derived. The

resulting physicalmaps(Fig. 2) were confirmed

byaseries of double digestions of dDNAlwith

different restriction enzymes. Four

representa-tive cleavagepatternsobtained are shown in Fig.

3. Each of them agreed withthe corresponding

double cleavage pattern predicted from the

physicalmaps(Fig.2).

(1-14x) (nx)

RightHand Terminrus

(1-14x)

(nx) ;

(nx) i

0.0 01 0.2 0.3 0,4 0.5 0.6 07 0.8 0:s1,0

0

0.1 0.2 0;3 04 0.5 0;6 0;7 0.8 0,9 1.0

FIG. 2. Scalemapsof KpnI, XhoI, PstI,and BamHIcleavagesitesontherepeatsequenceofHSV-1ANG dDNA1. The blackbarsrepresentthe550-base-pairinserts. nequalsthe numberof repeatsperunit-length moleculeofdDNAIand variesfrom9to17dependingonthe copy numberofinsertsperrepeat.

I

XhoI

PstI

Bam HI

VOL. 39,1981 77

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[image:3.493.73.430.342.616.2]

78 KAERNER ET AL.

C

0 - I(A

.c i a, m

s _coE ye sU. 4

_ x (A

4 - cc.

a _ E E C

a- 0. c c U)

_ _

a. a.

0 _

o c

x IC

_CL

E v

m

_ E

E-a. c

I

3.3x106d

Bam HI B BamHi D Pst C

_ Pst D

!36X106d

Bam Hi C

t

Kpn C

0.95x1O6d

0.72X106d

0.63X1O6d

_. _bCLc

cL vd

0

_C _

x cL

4.5X106d

t Xho B

Pst C _ Xho D

0.5X106d

I

2.5X106d

Kpn IC

Kpn D

Pst C

0.98X1O6d

Pst D

A B C D

FIG. 3. PstI-BamHI, BamHI-KpnI, XhoI-Pst,andPstI-Kpn I double digestions of HSV-1 ANG dDNA1.

Ethidiumbromide-stained restrictionfragments electrophoreticallyseparatedonagarosegels. The

nomen-clatureofthefragmentsisasdescribedfor Fig.1and2. The molecularweight calibrationwasperformed in

eachofthetwogels(AandB) by usingHindIII restrictionfragments ofX DNA(19), simian virus 40 DNA(15),

and HaeIII restriction fragments of4OX174RF DNA (10) as markers and is shown in gel A. For better

illustration thefragmentpatternsofthe PstI-BamHI and theBamHI-KpnI double digestswerecompared

with thefragmentpatternsof single digestswithPstI, BamHI, and KpnI in gel A. The fragment patterns of

thedoubledigestsin A and Bareshownschematicallyin C andD,respectively.

Forexample, the double digestion ofdDNAl

withPstI andBamHI created the left-hand

ter-minal fragment PstI-C, the fragments PstI-D

andBamHI-D,and the series ofright-hand

ter-minalfragmentsBamHI-B.Themajor step lad-der offragments in this digest had a base

frag-ment of 3.3 x 106 daltons, as expected, and

mappedbetween the BamHI cleavage site D-A

and the PstI cleavage site -A-D. BamHI-KpnI

digestion created the left-hand terminal

frag-ment BamHI-C and the series of right-hand

terminal fragments KpnI-C, together with the

otherfragments predicted fromthe maps shown

inFig.2.Similar conclusionscanbe drawn from

theXhoI-PstI andthePstI-KpnI cleavage

pat-terns.

Asshownin Fig.2, it isconceivable that the

550-base-pairsequenceinserts (1to 14copies [at

least]) cluster between the map positions of

about0.74and1.0;these map coordinatesfollow from the size of the left-hand terminal fragment

KpnI-B (3.1 x

10'

daltons) and the base

frag-ment of the right-hand terminal fragments

KpnI-C (1.6 x 10'daltons), assuming that the

latter containsatlast one of the inserts. In the mapsshown inFig. 2, the clusters of inserts are

tentatively placedatthe right-hand end of the

dDNAlmolecules, speculatingthat part of the insert sequence might function as a signal for

cutting the repeat concatemers to viral DNA

unitlength. Accordingtothe sequence

arrange-mentshowninFig. 2,all dDNA1 molecules had

m

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REPEAT UNITS OF DEFECTIVE HSV-1 ANG DNA

identical left-hand and right-hand ends. The

total sequence complexity of thedDNAlrepeat

unitwas calculated fromthephysical maps to

be about 6 x 106daltons (KpnI pattern, 6.0 x

106; BamHI pattem, 5.95 x 106; PstI pattern,

5.94 x 106;XhoIpattern,5.9 x 106 daltons).

Nonrandom distributionof the

550-base-pair insertions in the dDNAl repeat

se-quence units. The modelof the sequence

ar-rangementindDNAl (Fig. 2)raisesthequestion

of whether individual dDNAl molecules are

made up at random of repeats with different

copy numbers of the insertion sequence or

whethereach of thecompletedDNA1 molecules

consists ofacertain number of identical repeats,

each of which has the samenumber of inserts.

As an experimental approach to answer this question, dDNA1 was partially digested witha

series of restriction endonucleases, andthe

ob-tained fragment patternswereanalyzedon

aga-rosegels.Mostofthe enzymes assayedforthis

purpose rendered smears of fragment bands.

However, PstI endonuclease created partial

digestion fragment patterns which allowed a

conclusive interpretation. Figure4shows a

se-quential series of"snapshots" representing

ini-tialstatesof the PstIdigestion. Beside the

cre-ation of the final majorstepladder (0.35x 106

daltons[Fig.1]),twointermediate fragmentstep

ladders withstepsof0.7 x106andof 1 x 106to

1.1 x

106

daltons can be detected. The

corre-sponding base step fragments have molecular

weights of about11 x 106and 17 x 106,

respec-tively. Afurther group of bands which are not

clearly resolvedonthegelstartwith fragments

of about23x106daltons. Theinterpretationof

these results is shown in Fig. 5; the different

intermediate fragmentstep laddersapparently

represent theoriginationofdimers, trimers, and

ofmultimers ofahigherorderof therepeats. In

each of the multimers, the terminal0.94 x

106-dalton fragment PstI-D was cut off. The fact

that the step ladders of dimers and trimers,

which areclearlyresolved in the gelsshownin

Fig. 4,have stepsizes of0.7 x 106 (2 x 0.35 x

106) daltons and1x106(3x 0.35 x

10r)

daltons,

respectively, stronglysuggests that the

550-base-pair inserts are not interspersed atrandom in therepeatsofindividualdDNAl molecules. It is

conceivable that, in the case of random

distri-bution, all intermediate step ladders would

dis-play acommonstep size of0.35 x 106 daltons.

Thus, onemustconcludethat eachofthesteps

ofanindividualstep ladderindicates the

exist-ence of a certain class of dDNA1 molecules,

made up by identical repeats, each of which

containsaconstantnumberof inserts.This

im-plicates that different dDNAl molecules are

madeupby different numbers of repeats which

23s00 11,00

5,53

4,4

t

4,01

30 1,05 0,94

D C B A

FIG. 4. Partial digestion ofdDNA1 withPstI

re-striction endonuclease. Amounts (8 g) of dDNAI

weredigested with0.4Uof PstI (the definition of one

enzymeunitwasasgiven by the suppliers[BRLI).

Portionsof the digests containing 1.5 ug ofDNA were

takenfrom the reaction mixtureat 4(D),6(C), 8 (B),

and 12min (A) after thestartof the reaction and

wereanalyzedon anagarosegel. Thenumbers

rep-resent molecular weights x 106 of the fragments,

whichweredetermined by using HindIII fragments

ofXDNA, simian virus40 DNA, and HSV-1 ANG

viral standard DNAasmarkers. Filled arrows,

right-handterminalfragments(PstI-B,Fig.1);open arrow:

left-handterminalfragment (PstI-C,Fig.1).

areheterogeneousin size.Assuminganaverage

size of the complete molecules ofabout 100 x

106

daltons, therepeatnumbersmustvaryfrom

17 (1 insertperrepeat, 6x 106daltons)to9 (14

insertsperrepeat, 10.6x

10'

daltons).

Similardataon thecomposition ofrepetitive

defective HSV-1 (Justin) DNA has been

pro-videdbyFrenkeletal (5).Bypartial

denatura-tion studies, these authors could demonstrate

twotypes of DNAmoleculesbuilt up eitherby repeatunitswithoneadenine-plusthymine-rich

regionorbyrepeatunitswithtwoadenine-plus

thymine-rich regions.

79

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[image:5.493.251.447.61.379.2]

LeftHand Terminus

I.2 na

5.0

in c,

d

10.94

I l1e-a In I

41% o -( A 0n 0 I

IIIl

FII

i11.64a

i

1

17.93 iI

I

I

I I a r-Im we

5.7

0 I3

0.35'

18.98

0.7 _.

0.7

1.05 ',_

1.05

Final Stepladder Intermediate Stepladder Intermediate Stepladder Basis:5.0x10d Basis 3 10.94 x 106d Basis =16.88x106d

[image:6.493.53.447.45.321.2]

1Step:0.35x106d 1Step: 0.7x106d 1Step=1.05 x 106d

FIG. 5. Schematicalinterpretationofthepartialdigestion ofdDNAIwith PstI. The scheme shows three

differentdDNAI molecules with one, two, and three inserts per repeat. The insertsarerepresentedbyopen

bars.Preferentialcleavagesitesaremarkedbyarrows.The blackbars indicate thepreferentially originating

fragments.Thenumbersaremolecularweightsx106 DISCUSSION

A550-base-pairsequenceof theHSV-1ANG

genome wasfound earliertobeamplifiedtosix

copies in regions TRsand IRs of standard viral

DNA(9). Locker and Frenkel (11) andWagner

and Summers (21) have detected variable,

strain-specific sizeheterogeneitiesof the L ends

and the L-Sjoints of the DNA of HSV-1 strains

KOS, F, and Justin and have proposed that this

is duetotheamplificationofashortnucleotide

sequence. These authors, however, have

ana-lyzed viral DNAfrominfectedcells,whereas in

the study presented here, viralDNAextracted

frommaturevirionswasinvestigated. It should

be mentioned thatweobtainedthe sameresults

by using HSV ANG dDNAl isolated from

in-fectedcells.

Thedefective derivative HSV-1ANG dDNAl

is ofspecial interest as it originates from

non-contiguoussites of the parentalDNA.dDNA1,

besides beingpart ofTRs or IRS, including the

550-base-pair sequence, consists of sequences

from UL. Surprisingly, the 550-bapair

se-quence wasamplifiedtomuchhighercopy num-bers inthe dDNA1repeats than in the parental DNA. Thisfactisimportantinconnection with another result ofthe presentstudy which sug-gests that the totalpopulationofdDNAl

mole-cules consists of different homopolymers, each

of which is madeupof identical repeatswitha

constant copy number of the 550-base-pair

in-sertion sequence.Consideringresults of Locker

and Frenkel (12)whichsuggestthat individual

repeatunits of defective HSV-1 DNA are

elon-gatedtofull-size defective viralDNAbya

roll-ing-circlemechanism, it isconceivable that the

amplification of the insertion sequence in the

dDNAl repeatoccursbefore theonsetof

repli-cation.

Roizman (16) and Jacob etal. (8) have

pre-sented a hypothesis of the replication ofHSV

DNAviaarolling-circlemodeland the

genera-tion of all four isomers of HSV DNA from one

isomer. Onecentralfeature of this hypothesis is

the regeneration of the "a" sequence, which is

present at both ends of the molecule and is

reiterated in the L-Sjointin opposite

orienta-tion. Circularization of the DNA molecules by

ligationof cohesive ends would leadto theloss

ofonecopy of the "a" sequence whichhas to be

regeneratedto achievecompleteviralDNA

mol-ecules either before circularizationoraftertheir

excision from concatemeric DNA. The above

authorsproposedin their modelthatthe

regen-eration of the "a" sequence would occur after

replication via a rolling-circle mechanism and

that inthecourseof theregeneration of the "a"

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VO9%91REPEAT UNITS OF DEFECTIVE HSV-1 ANG DNA

sequence,theother isomers could beformned.

One conceivablespeculationfrom thepresent

datais that the550-base-pairsequencein

HSV-1 ANG standard DNA andindDNAl could be

(or could contain) the "a" sequence. The

ob-servedamplification of thissequence instandard

anddefective HSV-1 ANG DNA could then

re-flect one step of the viral DNA replication in

terms of the above-mentioned hypothesis,

which, forsomereason, doesnotstopafter the

regeneration of one copy of the "a" sequence.

Thefact that the550-base-pairsequenceis

am-plified to a higher degree in the repeats of

dDNA1 than in theparental DNAsuggeststhat

aplificationcanoccurin standard DNAaswell

asin the dDNAl repeatsthemselves, i.e., after

their formation from UL and the repeats from

the S component. On the other hand, higher

copy numbers of the amplified sequence are

evidently ofnoadvantage for the replication of

the repeats, as can be concluded from the

in-creasing number of insertions which correlate withadecreasing number of dDNA1molecules (Fig. 1).

Molecular cloning of dDNA1 restriction

en-zyme fragments and DNA sequencingare now

in progress to elucidate the sequence

arrange-mentofdDNA1. It should be mentionedfinally

that another type of defective HSV-1 ANG

DNA, dDNA2 (9) which closely resembles the

HD DNAderived from HSV-1 Justin by Frenkel

and co-workers (5, 6, 11), also shows multiple

insertions of the550-base-pairunit described in

thispaper(Kaerner,unpublisheddata).

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2. Bronson,D.L.,G. R.Dreesman,N.Biswal,and M.

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Hay-ward,H.Locker,and B.Roizman.1975.Anatomyof herpessimplexvirus DNA.III. Characterization of

de-fective DNA molecules and biological properties of

virus populations containing them. J. Virol.16:153-167. 6. Frenkel, N., H. Locker, W. Batterson, G. S. Hayward, and B. Roizman. 1976. Anatomy of herpes simplex virus DNA. VI. Defective DNAoriginatesfrom the S component. J. Virol. 20:527-531.

7.Graham,B.J., Z.Bengali,and G. F. Vande Woude. 1978. Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA. J.Virol. 25:878-887. 8. Jacob, R. J., L S. Morse, and B. Roizman. 1979. Anatomyof herpessimplex virus DNA. XII. Accumu-lation ofhead-to-tail concatemers in nuclei of infected cells and their role in the generationof the four isomeric arrangements of viral DNA. J. Virol.29:448-457. 9.Kaerner, H. C., L. B.Maichle, and C. H. Schrdder.

1979.Originof two different classes ofdefectiveHSV-1 AngelottiDNA.Nucleic Acids Res. 6:1467-1478. 10.Lee, A.S.,and R. LSinsheimer.1974. Acleavage map

ofbacteriophageOX174genome.Proc. Natl. Acad. Sci. U.S.A.71:2882-2886.

11. Locker,H., and N.Frenkel.1979.Structureand origin ofdefective genomes contained in serially passaged herpessimplexvirustype1(Justin).J.Virol. 29:1065-1077.

12.Locker,H., and N.Frenkel.1979.BamI,KpnI,andSall restriction enzyme maps of the DNAs ofherpes simplex virus strains Justin andF: occurrence of heterogeneities indefined regions of the viral DNA. J. Virol. 32:429-441.

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