Herpes simplex virus type 1 oriL is not required for virus replication or for the establishment and reactivation of latent infection in mice.

Vol.61, No. 11

Herpes Simplex Virus Type

1

oriL

Is

Not

Required for Virus

Replication

or

for the Establishment

and Reactivation of

Latent

Infection in Mice

MARYELLEN POLVINO-BODNAR, PAULO K. ORBERG,AND PRISCILLA A. SCHAFFER*

Laboratory of Tumor Virus Genetics, Dana-Farber CancerInstitute, andDepartment of Microbiology andMolecular

Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received 11May 1987/Accepted 31 July 1987

During thecourseofexperiments designed toisolate deletionmutantsofherpes simplexvirustype1 inthe geneencoding themajor DNA-binding protein, ICP8,amutant,d61, thatgrewefficiently in ICP8-expressing Vero cells butnotin normal Verocellswasisolated(P. K. Orberg andP. A.Schaffer, J.Virol.61:1136-1146,

1987). d61wasderived by cotransfection ofICP8-expressing Vero cells with infectious wild-type viral DNA and a plasmid, pDX, that contains an engineered 780-base-pair (bp) deletion in the ICP8 gene, as well as a

spontaneous -55-bp deletioninOriL. Gel electrophoresis and Southern blot analysisindicated thatd61 DNA

carried both deletionspresentinpDX. The ability of d61toreplicate despitethe deletion inOriL suggested that

afunctionalOriLisnotessential forvirusreplicationinvitro. Becaused61 harbored twomutations,asecond

mutant,ts+7, withadeletioninoriL-associated sequencesandanintact ICP8genewasconstructed. Bothd61 andts+7 replicated efficiently in their respective permissive host cells, althoughtheiryieldswereslightly lower

than those of control viruses with intact oriLsequences. An in vitro test oforigin function of isolated OriL sequences from wild-type virus and ts+7 showed that wild-typeOHiL, butnotts+7 OHIL, wasfunctional upon infection with helpervirus. Inaneffort todetermine therequirement for OriLinlatency, ts+7wascompared withwild-type virus for its abilitytoestablish, maintain, and be reactivatedfrom latent infection inamurine

eye model. The mutant was reactivated as efficiently as was wild-type virus from trigeminal ganglia after

cocultivation withpermissive cells, and each of thesevenreactivatedisolateswasshowntocarrythe-150-bp deletion characteristic ofts+7. These observations demonstratethatOHLis notrequiredfor virusreplicationin

vitroorfor theestablishment and reactivation of latent infectioninvivo.

The genomeof herpes simplex virus type 1 (HSV-1) is a

linear, double-stranded DNA molecule of approximately 160,000 base pairs (bp). It consists ofalong unique region

(UL) flanked by invertedrepeatsequencesaband b'a' andan

Scomponentconsisting ofashortunique region (Us) flanked

by the invertedrepeatsacand c'a'(Fig. 1, line 1). During the processof viral DNA synthesis, molecules aregenerated in which the long and short components of the genome are inverted relative to one another such that approximately equimolaramountsof the fourpossible isomers ofviral DNA

areproduced (1, 36). The viralgenomeisthoughttoreplicate byarolling-circle mechanism which yields large head-to-tail

concatemeric intermediates thataresubsequently cleavedto generate unit-length molecules (1, 3). Early electron micro-scopic studies provided evidence for the existence oftwo

origins of DNA synthesis in the HSV-1genome, one nearthe

centerofUL and another nearone end of the molecule (9, 14). Studies of thegenomesof defective interfering particles

generated during serialpassage of virus at high multiplicity of infection and tests of origin function with cloned viral DNAfragments have shown that HSV-1DNA contains two

copies of one origin, termed

oris,

located in the c and c'

invertedrepeats, and one copyofa second, more complex

origin, termed oriL, located near thecenter of UL (2, 7, 8, 16-18, 25, 35, 37, 43).

Fororigin function, the diploid

oris

requiresno morethan 90 bp, which includes an almost perfect palindromic

se-quenceof 45 bp (40).oriLshares considerablehomology with

oris

and contains a perfect 144-bp palindrome (12, 28, 45).

* Corresponding author.

While

oris

sequencesarestableuponcloning in bacteria, the larger oriL palindrome suffers deletions with high frequency when propagated inbacterial vectors(12, 45). Spontaneous or engineered deletions within

oris

and oriL palindromes result in loss of thecapacity forautonomous replication (38, 45).

The existence of two classes of defective interfering particles (class I, whose genomes contain

oris

sequences, and class II, whose genomes contain oriL sequences)

dem-onstratesthatbothoriginsareindeedcapableoffunctioning during productive infection in vitro (7, 8, 19). Little is known, however, about the requirement for each class of origin in the process of viral DNA synthesis (i.e., are oriL and bothcopies of

oris

essential for viral DNAsynthesis?)or

whether one class of origin is preferentially used during

latentinfection. The formerquestionwaspartially answered

inarecentreportthata mutantof HSV-1 lackingone copy of

oris

is viable(23). In thispaper,wedemonstrate thatthe

absence ofafunctional oriL has little effectonviral

replica-tion in vitro.Furthermore,weshow that oriL isnotrequired for the establishmentorreactivation of latent infection ina

murineeyemodel.

MATERIALS AND METHODS

Cells and viruses. African green monkey kidney (CV-1,

Vero, and U-47) and human embryonic lung (HEL) cells were propagated and maintained as previously described

(44). ICP8-expressing U-47 cells usedaspermissive hosts for the derivation and propagation of ICP8 deletion mutants

werederived by cotransfection ofVerocells with the

ICP8-3528 JOURNALOFVIROLOGY, Nov. 1987,p. 3528-3535

0022-538X/87/113528-08$02.00/0

CopyrightC 1987, American Society forMicrobiology

on November 10, 2019 by guest

http://jvi.asm.org/

a b UL b'a'o' US co

. , . I

0.34 0.35 0.36 0.37

3.3kb gB

5.6kb ? 2

--- ---- -- --

-1 I 1

0.38 0.39 0.40 0.41 0.42 0.43 0.44 4.2 kb IC P8 4.3kb D

0

lOkb ICP8 -.. 4.2kb DNAMI.

9kb ?

tsA24-I

4

BSB T 5

'W

1 'I

II I

XP K P TP S 6 pKEF-P4

i---p 7 pSGIS-SaIIA

8 pDX

B K

OxBomHI E E=EcoRI

---4 Kr-KpnI

s P-Pst I

--q

s SZSGII

TzBst E 11 Xz Xho I

i A

s x x

FIG. 1. Genomic location oforiLand

plasmids

used in this

study.

Theprototype arrangementof the HSV-1genomeis shown inline1.The

mapunits of the

expanded,

central

portion

ofthe

long

unique region

(UL)areshown inline2.The

transcripts

knowntomaptothis

region

of thegenome

(all

of the

early

or

0

kinetic class[15])andthelocation of theoriL (12, 45)areshowninline3. The

length

of the

transcript

is

indicated in kilobases

(kb).

The locationof the mutation in the ICP8ts mutantused in this

study,

tsA24, appearsin line4(44). Relevant

restrictionsitesand the

corresponding

nucleotide numbers(28)are

depicted

in thenextthreelines;the

symbol

Arepresentsdeletion inoriL sequencesof -55

bp

which ispresentin the

parental plasmid, pKEF-P4,

from which

pSG18-Sa/I

Aand

pDX

werederived.

containing plasmid pKEF-P4 (Fig.

1,

line

6)

and

pSV2neo

(27,

34).

TheKOS strain of HSV-1wasusedasthe

wild-type

virus

from which

temperature-sensitive

QsA24

[44])

anddeletion

mutantswerederived. Viruseswere

propagated

and

assayed

as

previously

described

(31).

Plasmids and

cloning.

Themaplocations of

oriL-associated

viralDNAinsertsin

plasmids

usedin this

study

areshownin

Fig.

1.

pKEF-P4

(4)

and

ptkCAToris

(not shown)

were

kindly

provided

by

N. DeLuca. The latter

plasmid

is a

derivative of

ptkCAT

(6)

which contains a

220-bp

SmaI

fragment

thatcontains

oris.

pSG18-Sall

A

(21;

Fig.

1,

line

7)

contains the intactgenefor ICP8anda

spontaneous

-55-bp

deletion in

oriL-

pDX (Fig.

1,

line

8)

was derived from

pSG18-SalI

A

by

digestion

withXhol and

religation.

pUC18

(46)

wasobtained from

Pharmacia,

Inc.

(Piscataway,

N.J.).

Restriction endonucleases and T4 DNA

ligase

were

ob-tained from New

England

BioLabs,

Inc.

(Beverly,

Mass.)

and usedas

suggested

by

the manufacturer.

Isolationof viral DNA.Infected-cellDNAwas

prepared

as

described

previously

(5).

After

proteinase

K

digestion,

viral

DNAwas

separated

from cellularDNA

by centrifugation

in

CsCl

gradients

(11).

KOS DNA was isolated from infected

HEL

cells; tsA24,

tsC,,

ts+3,

and

ts'7

DNAs wereisolated

from infected Vero

cells;

and d6l DNA was isolated from

infected U-47cells.

Isolation of viralDNA

fragments.

HSV DNA

fragments

for use as

probes

in Southern blot

hybridizations

wereisolated afterrestriction endonuclease

digestion

of

plasmid

DNAand

electrophoresis through

a 0.4% agarose

gel.

After

staining

withethidium

bromide,

bands of interestwere excised and

submitted to three

cycles

of

freezing

and

thawing

(32).

Agarose

was

separated

fromthe DNA solution

by

centrifu-gation.

ViralDNA

fragments

used intestsof

origin

function

were isolated after

digestion, electrophoresis

through

a4%

polyacrylamide

gel,

and electroelution into a

dialysis bag

(24).

Inallcases,DNA

fragments

wereethanol

precipitated

after

purification

by

passage

through

an

Elutip-d

column

(Schleicher

&

Schuell, Inc.,

Keene,

N.H.).

Blot

hybridization.

Specific

DNA sequences in

digests

of

viral or cellular DNA were detected

by

the method of

Southern

(33).

Probes were labeled with [12

P]dCTP

and

[32

P]dGTP

(Amersham

Corp., Arlington Heights,

111.)

by

nick translation

(24).

Marker transfer and marker rescue. Vero and U-47 cells

were cotransfected with infectious viral

(KOS

or

tsA24)

DNA and

plasmid (pDX

or

pKEF-P4)

DNAs as

previously

described

(29).

Cloning

and

sequencing0fiL

from

ts'7.

Viral DNA from

ts'7

was

digested

to

completion

with

KpnI

and cloned into

Kpnl-cleaved pUC18.

Clones

containing

the

Kpnl

V

frag-ment were identified

by

filter

hybridization

(13)

with the

KpnI

V

fragment

of

pKEF-P4

as the

probe.

The

Rsal-BamHl

fragment

of

Kpnl

V,

which contains the

large

palindrome

of

oriL,

wassubcloned intoBamHI-and

Hincll-cleaved

M13mp18.

The Rsal-BamHl

fragment

cloned into

M13mp18

wasthesamesizeon

acrylamide

gels (i.e.,

±5

bp)

astheunclonedRsaI-BamHI

fragment

derived

directly

from

viral

DNA,

indicating

thatfurther deletionhadnotoccurred

as aconsequence of

cloning

(data

not

shown).

Sequence analysis

wascarried out

using

thechain

termi-nation method of

Sanger

et al.

(30).

The Klenow

fragment

reactions were conducted at 50'C to avoid artifacts

gener-ated

by secondary

structures.The

products

were run on8%

polyacrylamide

gels containing

8 Mureaand 40% formam-ide.

Invitrotestsof

origin

function. The

procedure

usedtotest

whether

plasmid

DNAsequences

covalently

linkedto viral

origins

of

replication

were

amplified

aftertransfection and

infectionwith

helper

viruswassimilar tothatdescribed

by

other

investigators

(22, 39,

40,

45),

with one

significant

modification. Since

oriL

suffers deletions when

propagated

in bacteria

(12, 45),

gel-purified

viral DNA

fragments

were

ligated

tothetest

plasmid,

and the

ligation

mixturewasused

directly

in the transfection ofCV-1 cells.

The

gel-purified KpnI

V

fragment (map

units 0.406 to

0.419)

of KOS DNA and of

ts'7

DNA

(80

ng)

was

ligated

to

420 ng of

Kpnl-cleaved pUC18,

and 1/20 of the

ligation

mixture was

electrophoresed

in a 0.6%

gel

to

verify

that

ligation

had indeed occurred. One-half of the

remaining

C*j C-i 0 to-i it

to 0 Wm M

p 0 E In (Alf qt

CM C*j it) 102 in

i

I I I 11

'6

1 11 I I I 111-X X K T SP x XTK TB T SKE

p

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.612.147.460.61.247.2]

3530 POLVINO-BODNAR ET AL.

ligation

mixture or 110 ng of plasmid DNA (pUC18,

ptkCAToris,

orpDX)wascoprecipitatedwith salmon sperm

DNA,

and the

coprecipitate

was added to each of two

replicate

100-mmpetri dishescontaining2.5 x 106 CV-1cells

per dish. Four hours after

transfection,

cells were shocked

with15% glycerol for1min. At 10 hposttransfection, cells in

one of each

pair

of replicate dishes were infected with HSV-1

(KOS),

at amultiplicity of10PFU percell. At 16 h

postinfection,

all cells wereharvested andwashed, and total cellular DNAwasextractedby usingsodiumdodecyl sulfate and

proteinase

K

digestion,

phenol and chloroform

extrac-tion,

and ethanol

precipitation.

A 10-,ug portion of each

sample

was then

digested

with KpnI to excise unit-length

pUC18

from

replication

concatemersand withDpnI (which

digests input, methylated plasmid

DNA but not

newly

rep-licated, unmethylated

DNA

[20])

to

distinguish newly

repli-cated DNA. Control

samples containing

135 to 270 ng of

plasmid

DNA were also

digested

with KpnI alone or with

KpnI

plus

DpnI.All

samples

werethenelectrophoresedina

0.8% agarose

gel

and transferred to a nitrocellulose filter,

whichwas

probed

with32P-labeled pUC18, and an

autoradi-ogramwas obtained.

Latency testing.

Groups

of 12 7-week-old CD-1 mice

(Charles

River

Breeding Laboratories,

Inc.,

Kingston,

N.Y.)

wereinoculated with2 x 106PFUin20

p.l

of eitherwild-type

virus or

ts+7

after corneal scarification. On day3

postinoc-ulation,

eyeswabsweretaken from four miceandtrigeminal

ganglia

were removed from two mice for each virus (mice

usedforeye swabs mayor may nothavebeen the same as

those sacrificed for

ganglion

assays). Eye swabs were

as-sayed

directly

for infectious virus in Verocells, andganglia

were

frozen,

thawed,

sonicated,

clarifiedby low-speed

cen-trifugation,

and

assayed

in Vero cells. On day 30

postinoculation, surviving

micewere

sacrificed,

andganglia

were removed

immediately

(within 1 to 2minofdeath),cut

into

eight equal-sized pieces,

and cocultivated with Vero cells. On

day

5 of

cocultivation,

Vero cells and

ganglion

pieces

were

scraped

into

medium, frozen,

thawed, and

sonicated. The

suspension

was clarified

by

low-speed

cen-trifugation,

andthe supernatantfluidwas assayed for

infec-tious virusin Vero cell

monolayers.

RESULTS

Derivation of mutants ofHSV-1carrying deletions in OriL-associated sequences. (i) d61, a double mutant in ICP8 and

oriL.

Wehaveconstructedtwodeletionmutantsof HSV-1 in the gene

encoding

the

major

DNA-binding protein, ICP8,

andverifiedthatoneofthese, d61,hadincorporatedtheoriL

deletion,

aswellasthe ICP8deletionpresent in theplasmid

used in itsconstruction (27).

Briefly,

a780-bp deletionwas

introduced into

ICP8-coding

sequences ofplasmid

pSG18-Sall A to

yield pDX (Fig.

1, line 8). In addition to the

engineered

deletion,

pSG18-SaiI

A and consequently pDX also contain a spontaneous deletion in oriL-associated se-quences. Itwas assumedthat if oriL were essential for virus

replication, only

theICP8 deletion in pDX would be

incor-porated

into the viral genome when ICP8-expressing cells

were cotransfected with pDX and infectious, wild-type

DNA. Mutant d61 was isolated from theresulting progeny

on thebasisof itscapacitytogrow inICP8-expressing Vero

cells

(U-47 [27])

but notinnormal Verocells. Examination of

the DNAofd61revealed thatithadincorporated the 780-bp

engineered

deletionin theICP8gene (27). Unexpectedly, the

KpnI

V

fragment

of d61 (Fig. 2, lane 2) exhibited greater

mobility

than itsKOS counterpart did(lane 1). When KOS,

~~~~IIIL.

0 o o w

X Z;

V

I-e04

832bp >

780bp _{4 i}

KpnI KpnI/ BamHi Xho I

KpnI or

_L

0.406 A

armHi

Sal I

[image:3.612.370.515.63.413.2]

0.418

FIG. 2. Southern blot analysis of oriL-associated deletions in viral andplasmidDNAs.Viralorplasmid DNAs were digested with KpnIonly (leftmosttwolanes), KpnI and BamHI (next fourlanes),

orXhoI (rightmost lane), electrophoresed in an agarose gel, and transferredto anitrocellulose filter. Thefilter was then probed with theKpnI-SaIlfragment shownatthe bottomof the figure and inFig. 1, line5.On the leftmargin of thefigureareindicated thepositions of the oriL-containing KpnI V fragment and those of the KpnI-BamHI (0.406to0.411)fragments of KOS DNA (832 bp[28]) and of

d61, pKEF-P4, and pDX (780 bp). The size of the latter fragment

wasestimated bycomigration with the sequenced 780-bp (28)XhoI

fragmentof pKEF-P4, whichwasvisible in the rightmost lane of the ethidiumbromide-stainedgel but isnotdetected using thisprobe.

d61, pKEF-P4,andpDX DNAs were cleaved with KpnI and

BamHI and probed with the KpnI-SaII fragment shown at

the bottom of Fig. 2, the KpnI-BamHI fragments of d61,

pKEF-P4,andpDX exhibited greater mobility (780 bp)than

thecorresponding KOS fragment did (832 bp). These

obser-vations indicate that d61 had also incorporated the

sponta-neous oriL-associated deletion present in pDX and the

parental plasmid pSG18-SalI A. The efficient replicationof

d61 in ICP8-expressing cells suggested the possibility that

oriLmightnotberequiredfor viral replication in vitro. The

existence of multiple mutations in d61, however,

necessi-tated theisolation ofasecond mutant with a mutation inoriL only.

(ii) ts+7, a deletion mutant in OHL. A second mutant was

therefore constructedbymarker rescue of the ts mutation in theICP8mutant,tsA24,withplasmid pKEF-P4 (Fig. 1, line

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

B

Kp I Kp I

I a a

:.*X& ^

Kpn V

0.406

1ta11 lStall

J- )

FIG. 3. (A)KpnI restrictionpatternsofplasmid and viral DNAs. Theposition of the undeleted KpnIVfragment of KOS. tsA24. tsCl,

and ts+3 is shown onthe left. The arrow on the right side of the

figure indicates the position of the KpnI Vfragmentofts'7.ts.7 and ts+3werederivedfrom39°C plaques generated in the markerrescue

of tsA24withpKEF-P4. tsCt wasisolatedfrom 34°C plaques of the samemarkerrescue.(B) Southern blot analysis of the gel shown in

panelAprobed with the BstE IIfragment (shown in the diagramat

thebottom of thefigure) purified from pKEF-P4 and labeled with32P

bynicktranslation. Thediagram also illustrates the location of MriL within the KpnI V fragment relativetotheprobe.

6). pKEF-P4containsa55-bpdeletion intheoriL palindrome and an intact ICP8 gene (4, 45). On the basis of our

experience withd61, weanticipatedthatthe deletioninoriL

wouldbetransferredtothe viralgenomeconcomitantlywith therescueof the tsmutationby wild-type ICP8sequencesin

pKEF-P4. Of the 14

ts'

recombinants examined, one, ts47, wasfoundtocontainadeletion in theKpnIVfragment(Fig.

3). Unlike the deletion in the parental plasmid pKEF-P4, however, the deletion in ts+7 was -150bp, rather than 55

bp.Two otherplaqueisolateswerepickedfrom the progeny

of the marker transfer experiment: ts*3, from 39°C plates,

and tsC1, from 34°C plates. The DNA restriction patterns

and Southern blots shown in Fig. 3 clearly reflect the

existence of three classesof theKptIVfragment: undeleted

(KOS, tsA24, tsC1, and

ts`3),

containing a -55-bp deletion

(pKEF-P4 and d61), and containing a --150-bp deletion

(ts+7). Thus,tsC1 wasphenotypicallyandgenotypicallylike tsA24 (ts and

oriL'),

whereas ts+3 was phenotypically and

genotypically wildtype (ts+, oriL+)and ts+7 was

phenotyp-ically wild type

(ts')

and genotypically mutant (lacking

oriL).

Replication efficiency of oriL deletion mutants. The two

independently derived oriL mutants, ts 7 and d61, were

capable of efficient replication in one-cycle growth

experi-ments in their respective permissive cells (Table 1). Thus,

burst sizes ofts+7 and d61 were similar to thoseof control

viruses KOS and d21 in thetwocelltypes.The differencesin

the burst sizes of d21 and d61 relative to KOS and ts+7 probablyreflect therequirementforcomplementation of the

former mutant by the resident wild-type ICP8 gene in

permissive U-47 cells. Consistent with the data shown in Table1,the plaquesizes of KOS and d21 were slightly larger than those of ts47 and d61 in Vero and U-47 cells, respec-tively.

Testsof originfunctionofisolated viral DNAfragments.To

determinewhether the deletion inoriL-associated sequences

of ts+7 had affected origin function, we carried out in vitro origin function tests similar to those describedby others (22, 39, 40. 45). The distinguishing feature of the procedure employed in this study wasthat uncloned, gel-purified viral DNA fragments were used directly after ligation to test plasmidsequences. thus avoiding the possibility of introduc-ing additional deletions durintroduc-ing cloning in bacteria. As ex-pected, pUC18 sequences were not amplified in CV-1 cells after mock infection (Fig. 4, lane 1) or superinfection with HSV-1 (lane 2). The requirement for HSV-1-associated factors supplied in trans for

o5is

and(riL function is seen in lanes 3 and4(ptkCAToris) and lanes 5 and 6 (pUC18 ligated to the wild-type o0iL-containing KpnI V fragment), respec-tively. By contrast, pUC18 sequences ligated to the KpnI V fragment of ts#7 were not amplified after superinfection with HSV-1 (lane 8), nor was pDX, the plasmid used in the construction of d61 (lane 10). These tests also confirmed previous reports (45) that the oriLdeletion present in

pKEF-P4renders itincapable ofreplicating in experiments such as this (data not shown). These tests thus demonstrate that neither ts+7 nor pDX possesses an origin of viral DNA synthesis that can be driven by HSV-1 factors supplied in trans.

Sequencing of oriL deletions in d61 and ts+7. To further characterize the deletion in ts+7, we cloned the 309-bp RsaI-Ba,nHI fragment of the mutant in M13, sequenced it,

andcompared this sequence with that of thewild-type virus. The results of this analysis are shown in Fig. 5. Figure 5 presentsthe sequence of the 425-bp BstEII-BacmHIfragment of strain KOS (45), showingthe RsaI site at 114 to 118 bp, thelocation of the 144-bpinverted repeat, and the locations ofpertinent promoter regulatory sequences for the divergent

transcripts specifying the HSV major DNA-binding protein

and DNApolymerase (10, 15, 26, 41). The deletion in ts+7 is

148 bp in size and retains only one copy of the sequence CCAC found at positions 156 to 160 and 304to 308(Fig. 4). Thus, like other oriL-associated deletions described previ-ously, the ts 7 deletion appears to have occurred between shortrepeats (45). The deletion isasymmetric relative to the 144-bp inverted repeat, lacking 17 bp immediately to the right of the repeat and all but 13 bp of the 72-bp repeat

making up the left half of the palindrome. The deleted

sequences include the second distal signal upstream of the transcriptional start site of the major DNA-binding protein,

ICP8 (41), and may include as yet unidentified signals

upstream of the transcriptional start site and probably the

TABLE 1. Burst sizesof viruses containing intact ordeleted (IiL-associated sequences

Virus oril- ICP8 Burstsize"

sequences sequences (PFU/cell)

KOS Intact Intact 135

ts+7 Deleted Intact 96

d21 Intact Deleted 62

d61 Deleted Deleted 12

11Cells(106) wereinfectedat amultiplicityof 2.5 PFUJper cell(effective

multiplicity, IPFUpercell). washed, incubatedat37°Cfor 18h, harvested,

andassayed forinfectious virus. ICP8-deficient d21 and d61weretested in

ICP8-expressingU-47cells,andKOS andts-7weretested in Vero cells.

A

a.

a

a.%

i*

h

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.612.64.293.64.305.2]

3532 POLVINO-BODNAR ET AL.

"ATA" sequence of the DNApolymerasegene at 350 to 354

bp (10).

On the other hand, it is unlikely that the deletion includes any elements critical for transcription of either

essentialgeneasthemutantreplicates nearlyaswellasdoes

wild-type

virus

(Table

1).

Latency tests.

Having

established that ts+7 is replication

competent in cell

culture,

wenexttested therequirementfor functional oriL in the establishment of latency. For this purpose, groups of 7-week-old CD-1 mice were inoculated

with 2 x 106 PFU per eye of either wild-type virus orts+7

aftercorneal scarification. Eye swabs andtrigeminalganglia

were

assayed

directly

on Vero cells for infectious virus

during

acute infection

(day

3), and 12 ganglia from six survivingmiceper virusweretestedonday30by

cocultiva-tion for reactivacocultiva-tion of latent virus. The results of thesetests

demonstratethatts+7 behavedinvivo inamannersimilar to

wild-type

virus (Table 2). Thus ts+7was nearlyaslethal for

CD-1 miceaswas

wild-type

virus. Moreover, it

replicated

at

the site

of

inoculation and reached

trigeminal ganglia,

as

demonstrated

by

the presence of virus in eye swabs and

ganglia

on

day

3, andwasreactivated fromlatent infection as

efficiently

aswas

wild-type

virus. The DNAs of 7 of 12 virus

isolates reactivated from latent infection were tested by

Southern blot

analysis

for the presence of the deletion

characteristic ofts+7

(Fig.

6).

Compared

with twoisolates

derived

from mice inoculated with

wild-type virus,

allseven

isolates

derived from

ts+7-infected

mice exhibited the148-bp

deletion.We concludefromthesestudiesthatfunctionaloriL

is notrequiredfor the establishment orreactivationoflatent infection of mice.

DISCUSSION

The data

presented

herein demonstrate that oriL is not

required

for

growth

of

HSV

in vitroorfortheestablishment

and reactivation of latent infection inthe murineeye model.

These conclusions are based on observations made with

twodeletion mutantswhich

lack

afunctional

oriL.

The first

of the two mutants

isolated, d61,

contains mutations in

ICP8-coding

sequences,inoriL-associatedsequences, and in

severaladditional sites

(27).

Although unsuitable for

biolog-ical and molecular characterization because of its multiple

mutations,

the

replication

competence of d61 in

ICP8-expressing

cells

provided

the initial clue that HSV-1 can

replicate

inthe absence ofafunctionaloriL. Construction of

the second oriL deletion mutant, ts+7, provided the

neces-sarytooltoexaminethereplicationandlatencycompetence

of HSV-1

lacking

afunctional

oriL.

DNAsequenceanalysisandfunctionalcharacterization of

ts+7

revealed that the148-bp deletion (i)eliminates all but 11

bp

on the

right

end of the 144-bp oriL palindrome, (ii)

occurred between

direct

repeats, (iii) renders ts+7 oriL

unresponsive

to HSV-1 factors supplied in trans, (iv) has little effect on the growth properties of the mutant in vitro, and

(v)

has no detectable effect on the ability of ts+7 to

establish, maintain,

or bereactivated from latent infection. It is notable that during the construction of ts+7, the

148-bp

deletioninoriL-associated sequences was introduced

by using

a

plasmid,

pKEF-P4,thatcontains a 55-bp deletion

(45), demonstrating

that additional sequences can be lost in

mammaliancellsduringmarker transfer of such mutations to

the viralgenome.Of interestis theobservation that the large

deletion occurred between short direct repeats, as

previ-ously reported

for deletions generatedduring the cloning of

oriL

in

bacteria

(45). Whether the mechanism underlying

deletion of sequences between shortrepeats applies equally

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

,T4-4 ~~ .,

. 4

4.0 li,~~~~~~~~~~~~~

0

.*pDX

iptkCAToris

-pUC18

am

9o

I

a

4.*

MVY M V M V M V MVY

pUC18 ptkCAT- KOS ts' 7 pDX

oris

FIG. 4. Origin function test. Total cellular DNAs from CV-1

cells,transfectedwith the indicated DNAs(lanes 1 to10) andeither

mock infected(M)orinfected with KOS(V)orplasmidDNAs(lanes

11to14),weredigestedwithKpnIonly (lane 11)orwithKpnI plus

DpnI (all other lanes), electrophoresed in an agarose gel, and transferredtoanitrocellulosefilter.Thefilterwasthenprobedwith

32P-labeled pUC18. Lane 11containsamixture ofpDX, ptkCAT-oris, and pUC18. Lane 12contains pUC18 only, lane 13 contains

ptkCAToris only,andlane 14 shows pDXonly.

in bacterial and mammalian cells is unknown. Possible

mechanisms

leading

to deletions between short repeats

during cloning

inbacteria have been discussedby Weller et

al. (45).

Like all other deletedoriL clonessequencedto date (45) of

which the deletions lieat least partially withinthe inverted

repeat, that of ts+7 eliminated the entire palindrome.

Be-causenoneof these clones exhibitsorigin functionin assays

in

vitro,

the

palindrome

must contain elements requiredfor

origin

function. To date, detailed analysis of the cis-acting elements necessary for origin function has not been

re-ported,

and hence the specific role of the palindrome in

origin

function isnot known. Distinct homologies between the

highly

conserved (22) HSV-1 and HSV-2 oriL and

oris

palindromes

and papovavirus and adenovirus origins of DNA

replication

have been reported (18). Moreover, Elias et al.

(personal

communication) have reported that selected

palindromic

sequences of HSV-1 and HSV-2 oriL and

oris

likely

constitute thebindingsiteforaviral protein present in

infected cells.

Whatever theirrole in the initiationof viral DNA

replica-tion,

oriLsequenceshave also been postulated to play a role

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.327.557.62.389.2]

20 30 40 50 60 70 GCCGATGAAC CCCGGCGGCT GGCAACGCGG GGTCCCTGCG AGAGGCACAG ATGCTTACGG CGGCTACTTG GGGCCGCCGA CCGTTGCGCC CCAGGGACGC TCTCCGTGTC TACGAATGCC

DBPt

80 gp

TCAGGTGCTC CGGCCGGGT

AGTCCACGAG GCCCGGCCCA

1;0 Rsa1120 130 140 150 160 170 180 190 200

TGCGGTTGGT ATATGTACAC TTTACCTGGG GGCGTGCCGG ACCGCCCCAG CCCCTCfCAC ACCCCGCGCG TCATCA0CCG GTGG0CGTGG CCGCTATTAT ACGCCAACCA TATACATGTG AAATGGACCC CCGCACGGCC TGGCGGGGTC CGGGA GGTG TGGGCGCCGC AGTAGTCGGC CACCCGCACC GGCGATAATA

ts 7

210 220 230 240 250 260 270 280 290 300

AAAAAAAGTG AGAACGCGAA GCGTTCACTfT99 CCTAA TAATATATAT ATTATTAGGA CAAAGTGCGA ACGCTTCGCG TTCT CATTT TTTTATAATA

TTTTTTTCAC TCTTGCGCTT CGCAA CTCAAA CAGGATT ATTATATATA TAATAATCCT GTTTCACGCT TGCGAAGCGC AAGA rGAAA AAAATATTAT

pKEF-P4 TpoI I pKEF-P4

10 320 330 3140 350 360~ 370 1--10039040

GCGCAC CCACCGGCTA CGTCACGCTC CTGTCGGCCG CCGGCGGTCC ATAAGCCCGG CCGGCCGGGC CGACGCGAAT AAACCGGGCC GCCGGCCGGG CGCC G GGTGGCCGAT GCAGTGCGAG GACAGCCGGC GGCCGCCAGG TATTCGGGCC GGCCGGCCCG GCTGCGCTTA TTTGGCCCGG CGGCCGGCCC

ts*7

4410 420

GCGCCGCGCA GCAGCTCGCC GCCCGG

CGCGGCCCGT CGTCGACCGG CGGGCC

FIG. 5. Sequence of the 425-bpBstEII-BamHIfragmentcontainingoriL.The basesin bold print (positions 176to319)correspondtothe

144-bp invertedrepeat(45). Thesequencesin boxesarethe short 4-to6-bprepeatsbetween which deletions ints+7andpKEF-P4occurred,

respectively.The location of theRsaIsite (position114to118)usedtoclonethe RsaI-BamHIfragment ofts+7intoM13mpl8 for sequencing

purposesis alsoshown. The solid lines beneath bases 106to121, 137to152, and176to190representtheproximal, first, andsecond distal

signals, respectively, of the ICP8promoter(41).The location of the ATA box in the ICP8promoteris indicated by the dotted line (108to114).

Thetranscriptionalstartsiteof the ICP8geneis shownatposition 90. The probable ATA box and transcriptionalstartsitesofthepolymerase

gene areshownatpositions350to354 and 370 and 379, respectively (10).

in the transcriptional regulation of the two essential genes flanking oriL: those encoding the major DNA-binding

pro-tein, ICP8, and DNA polymerase (15). The second distal

signal of ICP8(41)(and possibly regulatory elements of the

polymerase gene [10]) is absent in ts+7, yet the virus

replicates nearly as efficiently as wild-type virus. As the

deletion in ts+7 is among the larger of the HSV-1 oriL

deletions tobe sequencedand thefirsttobeintroduced into

theviralgenomewith littleeffect on replication competence,

the role of palindromic sequences per se in regulation of

ICP8 andpol transcription during productive infection

re-mains unclear. The measurement ofactual levels of

tran-scription of these two genes in ts+7-infected cells is in

progress. Although site-directed mutagenesis will be

re-TABLE 2. Resultsoflatency testsin CD-1 mice

Virus titer inspecimens

Virus Lethalitya Eye Latent

ViruLethalit

swabs"

Gangliac infectiond

Wildtype 9/26 (35) 1.2 x 102 1.5 x 104 12/12

3.7 x 102 2.2 x 104

4.2x 102 4.7 x 104

1.8 x 103 5.4 x 104

tsI7 3/10(30) 1.0 x 101 2.3 x 104 10/12

3.1 X 102 2.9 x 104

7.1 x 102 5.8 x 104

1.1X 1013 6.2 x104

aLethalityispresentedastheratio of the number of dead animalstothe

number of animals inoculatedwith2x 106PFUper eye.

bEye swabsweretaken from fourmiceonday3postinoculation. Swabs from both eyes of eachmouse werepooledandassayedonVerocells.Titers aretotalPFU ineye swabsuspensions.

cTwo miceweresacrificedonday3postinoculation,andindividualganglia were homogenized and plated directly on Verocells. Titers arePFU per ganglion.

dOn day 30 postinoculation, ganglia were removed from six mice (12 ganglia) and assayed for reactivatable virusbycocultivationwith Verocells.

Noinfectious viruswasdetectedin sixtrigeminalgangliaofmice inoculated withwild-typevirus, removedonday 30,homogenized,andplateddirectly.

KOS ts 7

1 2 12 3 4 5 6

.p -

-In N 0 +

7 X

4e-Om __m am_ _ _ _ R

w v

FIG. 6. Southern blot of DNAs of virus isolatesobtained from

trigeminal gangliaofmiceinoculated withwild-type virusorts+7.

Two isolates from KOS-infected mice were compared with seven

isolates from ts+7-infected mice. Total DNAs from cells infected with each isolatewerecleaved withBamHI, electrophoresedon an agarosegel, and transferred toa nitrocellulosefilter, andthe filter wasprobedwith theKpnIV fragmentwhich hybridizestoboththe BamHIR and Vfragments.ControlKOS andts+7DNAsareshown

in theright-handtwolanes. 10

ACCACGGGGT TCCTGCCCCA

100 GCGTCTGATA CGCAGACTAT

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.612.58.553.67.242.2] [image:6.612.345.519.322.632.2] [image:6.612.55.295.496.620.2]

3534 POLVINO-BODNAR ET AL.

quiired

to elucidate the

specific

roles of

oriL

sequences in

origin function and transcriptional regulation, theavailability

ofanisogenicseries of sequenced deletion mutationsinoriL should prove useful, with the unfortunate caveat that their

introduction into the viral genome by marker transfer may

result in further sequence loss.

The data

presented

herein demonstrate that

oriL

is not

required for virus replication in vitro, implying that two

copies of

oris

are sufficient for this purpose. In a recent

report, Longnecker and Roizman reported the viability of a

mutant of HSV-1 strain F, R7023, that contains oriL and only one copy of

oris

(23). The viability ofthis mutant

demon-stratesthat the two remaining origins (one copy of

oris

and

oriL) are sufficient for virus replication. The observations

that viable

mut4nts

of HSV-1 may lackeither oriL or one of

two copies of

oris

raise interesting questions regarding the

role(s)of the two types of origin in thebiology of HSV-1. (i)

Iseither type of origin preferentially used during the lytic or

latent modes of infection, and (ii) why have both been

conserved in the HSV-1 and HSV-2 genomes?

With regard tothe former question, we have demonstrated

the replication and latency competence of

ts+7

in

vivo,

implyingthat anintact oriL isnot requiredfor the

establish-ment, maintenance, or reactivation of latency. The only

other virus for which a latency-specific origin has been

suggested is Epstein-Barr virus, in which oriP has been

shown to function during latent infection (42, 47). In this

system, however, no origins other than oriP have yet been

identified (although they may exist), and it has not been

shown thatoriP does not also function during lytic infection.

Some insightinto the secondquestion mightbegained by

comparisonofHSVwithotherherpesviruses.Inthis regard,

it is notablethat the genome of varicella-zoster virus, a virus

which shares extensive biological and DNA structural

simi-larities with HSV, contains a diploid

oris

but lacks an oriL

equivalent (39).

ACKNOWLEDGMENTS

WethankN.DeLuca forvaluable discussions, M. Bush, D. Coen, J. Jacobson, D. Knipe, K. Tyler, E. Villareal, and D. Yager for assistance in latency tests of ts+7, and M. Cook for manuscript preparation.

This investigation was supported by Public Health Service

Pro-gramProject grant no. CA21082 from the National Cancer Institute and grant no. A124010 from the National Institute of Allergy and Infectious Diseases. M.P.-B. is supported by a Special Fellowship fromthe Leukemia SocietyofAmerica. P.K.O. was supported by Postdoctoral Fellowship no. DRG-840 from the Damon Runyon-WalterWinchell Cancer Fund.

LITERATURE CITED

1. Ben-Porat,T.1982.Organization and replication of herpesvirus DNA, p. 147-172. In A. S. Kaplan (ed.), Organization and replicationofviral DNA. CRC Press, Inc., Boca Raton, Fla.

2. Cuifo, D.M., and G. S. Hayward. 1981. Tandem repeat

defec-tive DNA fromthe L segment of the HSV genome, p. 107-128.

In Y. Becker (ed.), Herpesvirus DNA. Martinus Nijhoff, The Hague, The Netherlands.

3. Deiss, L. P., and N. Frenkel. 1986. Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging

and involves amplification of the terminally reiterated a se-quence.J. Virol. 57:933-941.

4. DeLuca, N., D. Bzik, V. C. Bond, S. Person, and W. Snipes. 1982. Nucleotide sequences of herpes simplex virus type 1 (HSV-1) affecting virus entry, cell fusion, and production of glycoprotein gB (VP7). Virology 122:411-423.

5. DeLuca, N. A., M. A. Courtney, and P. A. Schaffer. 1984.

Temperature-sensitive mutants in herpes simplex virus type 1

ICP4 permissive for early gene expression. J. Virol. 52:767-776. 6. DeLuca, N. A., and P. A. Schaffer. 1985. Activation of immedi-ate-early, early, and late promoters by temperature-sensitive

and wild-type forms of herpes simplex virus type 1 protein

ICP4. Mol. Cell. Biol. 5:1997-2008.

7. Frenkel, N.1981.Defectiveinterferingherpesviruses,p. 91-120. In A. J. Nahmias, W. R. Dowdle, and R. F. Schinazi(ed.), The human herpesviruses. Elsevier, New York.

8. Frenkel, N.,

H.

Locker, and D. A.

Vlazny.

1980. Studies of defective herpes simplex viruses. Ann. N.Y. Acad. Sci. 354:347-370.

9. Friedmann, A., J. Shlomai, and Y. Becker. 1977. Electron microscopy of herpes simplex virus DNA molecules isolated from infected cells by centrifugation in CsCl density gradients.

J. Gen. Virol. 34:507-522.

10. Gibbs, J. S., H. C. Chiou, J. D. Hall, D. W. Mount, M. J.

Retondo, S. K. Weller, and D. M. Coen. 1985. Sequence and mapping

analyses

of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain. Proc. Natl. Acad. Sci. USA 82:7969-7973.

11. Goldin, A. L., R. M. Sandri-Goldin, M. Levine, and J. C.

Glorioso. 1981. Cloning of herpes simplex virus type 1

se-quences representing the whole genome. J. Virol. 38:50-58. 12. Gray, C. P., and H. C.Kaerner. 1984. Sequence of the putative

origin of replication in the UL region of herpes simplex virus type 1 ANG DNA. J. Gen. Virol. 65:2109-2119.

13. Grunstein, M., and D. Hogness. 1975. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific

gene. Proc. Natl. Acad. Sci. USA 72:3961-3965.

14. Hirsch, I., G. Cabral, M. Patterson, and N. Biswal. 1977. Studies

on the intracellular replicating DNA of herpes simplex virus type 1. Virology 81:48-61.

15. Holland, L. E., R. M. Sandri-Goldin, A. L. Goldin, J. C. Glorioso, and M. Levine. 1984. Transcriptional and genetic analyses of the herpes simplex virus type 1 genome: coordinates 0.29 to 0.45. J. Virol. 49:947-959.

16. Kaerner, H. C., I. B.Maichle,A. Ott, and C. H.

Schroder.

1979. Origin of two different classes of defective HSV-1 Angelotti DNA. Nucleic Acids Res. 6:1467-1478.

17. Kaerner, H. C., A. Ott-Hartmann, R.Schatten, C. H.

Schroder,

and C. P. Gray. 1981. Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus

type 1 Angelotti DNA. J. Virol. 39:75-81.

18. Knopf, C. W., B. Spies, and H. C. Kaerner. 1986. The DNA replication origins of herpes simplex virus type 1 strain Angelotti. Nucleic Acids Res. 14:8655-8667.

19. Knopf, C. W., G. Strauss, A. Ott-Harman, R. Schatten, and H. C. Kaerner. 1983. Herpes simplex virus defective genomes: structure of HSV-1 ANG defective DNA of classII and encoded polypeptides. J. Gen. Virol. 64:2455-2470.

20. Lacks, S., and B. Greenberg. 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114:153-168. 21. Lee, C. K., and D. M. Knipe. 1983. Thermolabile in vivo

DNA-binding activity associated with a protein encoded by mutants of herpes simplex virus type 1. J. Virol. 46:909-919.

22. Lockshon, D., and D. A. Galloway. 1986. Cloning and

charac-terization oforiL2,a large palindromic DNA replication origin of herpes simplex virus type 2. J. Virol. 58:513-521.

23. Longnecker, R., and B. Roizman. 1986. Generation of an

invert-ing herpes simplex virus 1 mutant lackinvert-ing the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the

a47

gene. J. Virol. 58:583-591.

24. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular

cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

25. Mocarski, E. S., and B. Roizman. 1982. Herpesvirus-dependent amplification and inversion of cell-associated viral thymidine

kinase gene flanked by viral a sequences and linked to an origin of viral DNA replication. Proc. Natl. Acad. Sci. USA

79:5626-5630.

26. Morse, L. S., L. Pereira, B. Roizman, and P. A. Schaffer. 1978. J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

Anatomyof herpes simplex virus (HSV) DNA. X. Mapping of viral genes by analysis of polypeptides and functions specified by HSV-1 x HSV-2 recombinants. J. Virol. 26:389-410. 27. Orberg, P. K., and P. A. Schaffer. 1987. Expression of herpes

simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition ofwild-type virus. J. Virol. 61:1136-1146.

28. Quinn, J. P., and D. J. McGeoch. 1985. DNA sequence of the region in the genome of herpes simplex virus type 1 containing the genes for DNA polymerase and DNA binding protein. Nucleic Acids Res. 13:8143-8163.

29. Sacks, W. R., C. C.Greene, D. P. Aschman,and P. A. Schaffer.

1985. Herpes simplex virus type1 ICP27 isanessential regula-toryprotein. J. Virol. 55:796-805.

30. Sanger, F., S. Nicklen, and A. Coulsen. 1977. DNAsequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

31. Schaffer, P. A., V. C. Carter, and M. C. Timbury. 1978.

Collaborativecomplementation study of temperature-sensitive

mutants of herpes simplex virus types 1 and 2. J. Virol. 27:490-504.

32. Smith, H. 0. 1980. Recovery of DNA from gels. Methods Enzymol. 65:371-380.

33. Southern, E. M. 1975. Detection ofspecific sequences among

DNAfragments separated by gel electrophoresis. J. Mol. Biol.

98:503-517.

34. Southern, P.J.,and P.Berg. 1982.Transformation of

mamma-lian cells to antibiotic resistance with a bacterial gene under controlof the SV40 early region promoter. J. Mol. Appl. Genet. 1:327-341.

35. Spaete, R.R.,and N. Frenkel. 1985.The herpessimplex virus amplicon: analyses of cis-acting replication functions. Proc.

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

36. Spear,P.G.,and B. Roizman.1980.Herpessimplexviruses, p. 615-745. In J. Tooze (ed.), DNA tumor viruses. Cold Spring HarborLaboratory, ColdSpring Harbor,N.Y.

37. Stow,N. D. 1982. Localization ofanorigin ofDNAreplication

within theTRsIIRs repeatedregion of the herpes simplex virus type 1 genome. EMBO J. 1:863-867.

38. Stow, N. D. 1985. Mutagenesis of a herpes simplex virus origin of DNA replication and its effect on viral interference. J. Gen. Virol. 66:31-42.

39. Stow,N.D., andA. Davison. 1986. Identification ofa varicella-zoster virus origin of DNA replication and its activation by herpes simplex virus type 1 gene products. J. Gen. Virol.

67:1613-1623.

40. Stow, N., and E. C. McMonagle. 1983. Characterization ofthe

TRsIIRsorigin of DNA replication of herpes simplex virus type

1. Virology 130:427-438.

41. Su, L., and D. M.Knipe. 1987. Mapping of thetranscriptional initiation site of the herpes simplex virus type 1 ICP8 gene in infected and transfected cells. J. Virol. 61:615-620.

42. Sugden, B., K. Marsh, and J. Yates. 1985. A vector that

replicates as a plasmid and can be efficiently selected in

B-lymphoblasts transformed by Epstein-Barr virus. Mol. Cell. Biol. 5:410-413.

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

44. Weller, S. K., K. J. Lee, D. J. Sabourin, and P. A. Schaffer.

1983.Genetic analysis oftemperature-sensitive mutantswhich define the gene for the major herpes simplex virus type 1

DNA-bindingprotein. J.Virol.45:354-366.

45. Weller,S.K.,A.Spadaro, J.E.Schaffer,A. W.Murray,A. M.

Maxam, and P. A. Schaffer. 1985. Cloning, sequencing, and functionalanalysis of oriL,aherpessimplex virus type1origin ofDNAsynthesis. Mol. Cell. Biol. 5:930-942.

46. Yanisch-Perron, C., J. Vierira,andJ. Messing. 1985.Improved

M13 phage cloning vectors and host strains: nucleotide

se-quences ofthe M13mpl8 and pUC19 vectors. Gene

33:103-119.

47. Yates, J. L.,N.Warren,and B.Sugden.1985.Stablereplication ofplasmids derived fromEpstein-Barr virus in various

mamma-liancells. Nature(London) 313:812-815.

on November 10, 2019 by guest

http://jvi.asm.org/