A cell-free recombination system for site-specific integration of multigenic shuttle plasmids into the herpes simplex virus type 1 genome.

JOURNAL OF VIROLOGY, Sept. 1992, p. 5509-5515 0022-538X/92/095509-07$02.00/0

CopyrightX 1992, American Society forMicrobiology

A

Cell-Free

Recombination

System for Site-Specific

Integration

of

Multigenic Shuttle

Plasmids

into the

Herpes

Simplex Virus

Type

1 Genome

PHILIPJ. GAGE,' BRIANSAUER,2 MYRON LEVINE,3ANDJOSEPH C. GLORIOSO4* Department of Microbiology and Immunology, Universityof Michigan MedicalSchool, Ann Arbor, Michigan

48109-06201;

Experimental Station, E328, Dupont-Merck PharmaceuticalCompany, Wilmington,

Delaware 19880-0328;DepartmentofHuman Genetics, University of Michigan MedicalSchool,

AnnArbor, Michigan 48109-06183; and Department of MolecularGenetics andBiochemistry

University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania152614

Received 23 April1992/Accepted 19 June 1992

Thisreport describesanovel method for complementation studies ofdefective herpes simplexvirus (HSV)

genes.Viraltestgeneand nonviral reportergenecassetteswererapidlyintegrated intotheHSVgenomeina

site-specific and reversible manner by using the P1 phage-based Cre-ox recombination system. Shuttle plasmids containedafunctionalloxP recombination site,anexpressible form ofthe bacterial lacZgene,anda

copyofthe

wild-type

glycoprotein B (gB) gene ordouble mutant gB allele containing both a

temperature-sensitive (ts) mutation and a syncytium (syn)-forming mutation. A recipient viralgenome, KAT:dloxl, was

constructed from the HSV tpe 1 (syn) gB-deficient mutant virus, KAT, by marker transfer of the loxP

recombination site into the viral thymidine kinase locus. Shuttle plasmids ofup to 12.9 kb in length were

recombined with high efficiency(11to20%o)into theKAT::laxl genomein cell-free, Cre-mediated recombi-nation reactions. Expression ofa functional wild-type ordouble mutant gB polypeptide complementedthe nonfunctional polypeptide expressed from the deleted, normal gB locus and allowed production ofeither wild-type orSyn- plaques onVero cells. The latter recombinant viruswasalsotsfor growth. Theability to

express viral genes from plasmids which can be shuttled into and out of the HSV genome in cell-free

recombination reactions makes thisapowerfulmethodfor performinggenetic studies of thebiologicproperties of viralgeneproducts.

Herpes simplex virus type1 (HSV-1) belongstoafamily

of animal viruses typified by having large, complex DNA

genomes (100 to 250 kb) (for a review, see reference 25).

HSV-1 contains approximately 72 genes encodingproteins

which maybe classified as providing either essential

func-tions for infectious-particle production in cell culture or accessoryfunctions which contribute toviral pathogenesis and extend the virushostcellrangeduring natural infections

in vivo.Although the entire HSV genomicsequenceisnow

known (21-23), the biological properties and roles in repli-cation formany ofthese viral functions have notyetbeen elucidated.

Theproduction and characterizationof HSVmutantshave provided the central approachtouncoveringthe functional roles of individualgenes incarryingout differentphasesof thecomplexvirus lifecycle.Theprincipal strategyhas been to genetically alter cloned viral DNA segments and then study the affected biologicfeatures of the mutantproducts eitherintransient transfection experiments orin infections after marker transfer of the mutant gene into the viral

genomeby homologous recombination. While transient as-sayscanbe useful for the initial characterization ofmutant constructs,manyexperimental approaches requirethat mu-tantgenesbe transferred into the viralgenome (3, 7, 9, 11, 12, 29)foranalysisoftheirimpactincomplex settingssuch as infection of cell cultures or animals. Genetic studies whichrelyonmarker transferproceduresareusuallyslowed by the requirementforpriorcharacterization ofthe mutant

* Correspondingauthor.

constructand bya lowyield of recombinants. Asa

conse-quence, this approach is rather laborious, particularly for experiments requiring the analysis of large panels ofmutant constructs. Moreover, sequences transferred into the viral genome by homologous recombination are not directly

re-coverable butmustbe recloned priortofurther characteri-zation or manipulation. Amorerapid method for

introduc-tion of mutant constructs into the HSV genome, which would permit the identification of relevant mutations that resultinaparticular phenotype, would greatlyfacilitate the geneticandbiochemicalcharacterization of these viralgenes

and theirproducts.

Workby Saueretal.(28) demonstratedthatplasmidDNA couldbeefficientlyinserted intoaherpesvirusgenomeinan

in vitrorecombination reaction.By taking advantageof the Cre-lox site-specific recombination machinery of bacterio-phage P1 (2, 16, 33), a plasmid containing the 34-bp loxP recombination site, but completely lacking in homologous herpesvirus sequences, could be specifically inserted at a

loxP site engineered into the pseudorabies virus genome. Theinserted plasmid disruptedthe nonessential gIII glyco-protein gene. Recombination was efficient and could be accomplished in a cell-free reaction mixture consisting of onlythe viral andplasmidDNAsandthe Cre recombinase. Transfectionof the reactionmixture into suitableeukaryotic cells allowedfor therescueofinfectious recombinantvirus.

Recombinationefficiencies ofatleast8%werereported, and

the inserted plasmids were stably maintained in the viral

genome.Furthermore,because the Crerecombination

reac-tion is reversible, the inserted shuttle plasmid could be

recovered from the recombinant viral genome by a Cre-5509

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5510 GAGE ET AL.

mediated intramolecular recombination reaction and

by

propagation aftertransformation ofEscherichia coli.

Inthis report,wedescribehowthe Cre-lox shuttlevector strategy was adapted for complementation of an HSV-1

mutantwiththe essential glycoprotein B(gB)gene deleted.

Wild-typeand mutantcopies of thegBgene were

efficiently

shuttled into andout ofagB- loxP recipient virusgenome. Recombinant viruses were identified on the basis of their

blue-plaque phenotype derived from expression of a lacZ

reporter gene present within the inserted shuttle

plasmid.

Expression offunctional gB polypeptides from the inserted plasmids complemented the gB- defect of the

recipient

virus. Insertion of a temperature-sensitive (ts) and syn

double mutant gBalleleconferred bothmutant phenotypes

on therecombinant virus. These studies provided evidence that the Cre-loxshuttle systemwill beuseful for thegenetic

analysisof the rolesofboth viralgenesaswellas cis-acting genomicelements whichfunction during virusreplication.

MATERIALS AND METHODS

Cellsandvirusstrains. Verocells andthe D6cell linewere maintained in Eagle's minimum essential medium (MEM) (GIBCO Laboratories, Gaithersburg, Md.) supplemented

with 10 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), the nonessential amino acids, and 5% newborncalfserum (GIBCO Laboratories). D6 cells, which were previously derived by transforming Vero cells with

pKBXX (8) and pSV2neo (31), express HSV gB-1 upon infectionandcomplement the growth of gB- viruses (8). D6 cellsweremaintained inculturemedium supplementedwith 200jigof the antibioticG418(GIBCO Laboratories) perml. For transfection experiments, the cells were maintained in MEM supplemented with 5% fetal calf serum (GIBCO

Laboratories) instead of newborn calfserum.

KAT is a derivative of HSV-1 (strain KOS) carrying a

969-bp BstEII deletion within the essential gB gene and

expresses an internally deleted, nonfunctional gB

polypep-tide which is secreted into the medium (8). Both KAT and thecomplementingcellline, D6,werekindlyprovided by S. Person. KATandKAT::loxl (described below)were

propa-gated, andtheirtiters onD6 cellsweredeterminedat 370C. Cre-mediated recombinants derived from KAT::loxl were firstisolatedonD6cells and thengrown, and their titerson

Verocellsweredetermined at37°C. Recombinants contain-ing the ts form of gB were tested for this phenotype by plaqueassaysonVero cellsat34 and39°C. Allrecombinant virusstockswerederived fromplaque-purifiedisolates.

For plaque assays orindividual plaque isolations, serial dilutions of virus stocks were inoculated onto confluent monolayers of the indicated cell line which were

subse-quentlyincubatedunder methylcellulose (0.5% methylcellu-lose in MEM supplemented with 10 mM HEPES and 2% newborn calfserum) at 370C until plaques developed. The

cultures were then stained either with crystal violet (1%

crystal violetin 50% ethanol)orwithBluo-gal (halogenated

indolyl-13-D-galactoside) for ,B-galactosidase (,3-gal) activity

(see below), and when appropriate, virus from individual blue plaqueswasisolated for subsequentpurification.

Bacterialstrains.Plasmidswereisolatedandpropagated in

E. colistrainsDH5a (15)orGM119 (giftfromD. Oxender).

Plasmid-transformed bacteriaweregrowninLuria broth(10

goftryptoneperliter, 5gofyeast extractperliter, 171mM

NaCl) supplemented with 75 ,ug of ampicillin (Boehringer MannheimBiochemicals, Indianapolis, Ind.)permlor onL

agarplates containing ampicillin.

Construction of plasmids. All

cloning

steps and

plasmid

propagations were

performed

by

standard

procedures

(15,

17,

27).

Enzymeswere

purchased

from

GIBCO-BRL,

Inc.,

or

Boehringer

Mannheim Biochemicals and usedas

specified

by the manufacturers.

pLink760

(34),

pON1

(32), pKBXX

(8), and pBS64 and pBS65

(28)

have all been described previously. pUCXI

(19)

contains the HSV-1 BamHI P

frag-ment

containing

the

complete

thymidine

kinase

(tk)

locus subcloned from

pXl

(18)

into

pUC9.

pJG101

was

con-structed from

pUCX1

by inserting

a

354-bp

NaeI-SmaI

fragment

containing

a functional

loxP

site from

pBS64

into

theuniqueSnaBI site within the coding sequence for the tk gene. This354-bp insertionoccursbetween residues 110and

111 of the TK protein and was

expected

to

abrogate

TK

activity. A 270-bp

BamHI-PvuII

fragment from

pBS65,

includinga functional laxPsite, wasinserted into

pBSM13-(Stratagene,

La

Jolla,

Calif.)

betweentheBamHI andSmaI sites, creating

pBSloxP. pIEPlacZ

was constructed

by

sub-cloning a 763-bp

BglII

fragment from pLink760 into the unique

BglII

site of

pON1,

placing a promoterless cassette

containing the bacterial lacZ gene under the control ofthe human cytomegalovirus immediate-early promoter

(HCM

VIEP). The HCMVIEP-lacZ gene cassette from

pIEPlacZ

was inserted at the unique BamHI site of pBSloxP as a

4.7-kb BamHI fragment, creating

pJG103.

Finally, to

con-struct pJG108 (see Fig. 3),

pJG103

was partially digested withBamHI, the linear unit-length plasmid

fragment

was gel purified, anda3.7-kb HSV-1

BamHI-BclI

fragment contain-ing the entire gB-1 (strain KOS) gene from pKBXX was inserted in the orientation shown (see Fig. 3).

pJG102

(see Fig. 3) was constructed in a similar fashion frompBSloxP by inserting (i) the ts syn

gB-1

allele from the HSV-1 mutant tsB5 asa 4.9-kb

KpnI

fragment (0.346 to 0.377 map units) at

the pBSloxP

KpnI

site and (ii) the 4.7-kb

BamHI

HCM VIEP-lacZ gene cassette from pIEPlacZ at the pBSloxP BamHI site.

Viral DNA isolation. ViralDNA wasisolated by a modifi-cation of standard methods for high-molecular-weight eu-karyotic DNA isolation (4, 14). Confluent Vero cell

mono-layers were infected ata multiplicity of infection of 5. At 24 h postinfection, virions free in the medium and released by freeze-thawing cells were combined and pelleted at 75,000x g for 40 min. The virion pellet was resuspended in lysis

solution (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.6% sodium dodecyl sulfate, and 0.25 mg of proteinase K per ml)

and incubated at

37°C

for 8 to 12 h. DNA was multiply extracted first with phenol-chloroform (1:1) and then with chloroform, precipitated with isopropanol, and resuspended in TE (10 mMTris-HCl [pH 8.0], 1 mM EDTA) buffer.

Marker transfer ofloxP into KAT. TheloxPsite in

pJG101

was marker transferred into the tk locus of the KAT genome by homologous recombination after cotransfection of viral

and plasmid DNAs into subconfluent D6 cells as described by Graham and van der Eb (13) and modified by Homa et al. (18). Because marker transfer of the loxP fragment would

interrupt TK expression, transfection lysates were enriched for TK- viruses (24, 26) by serial propagation at low

multiplicity of infection (<0.001) on D6 cells in MEM supplemented with 100

jig

of thymine

1-,B-D-arabinofurano-side (araT) (Sigma Chemicals, St. Louis, Mo.) per ml. The enriched TK- stock was used to inoculate D6 cell monolay-ers, individual plaques were isolated, and small virus stocks were grown on D6 cells in microtiter plates. Infected cell lysates were tested for theloxPinsert by DNA dot blot assay with theNaeI-SmaI loxP fragment from pBS64 as probe.A recombinant virus,

KAT::loxl,

containing the

loxP

insert,

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INSERTION OF SHUITLE PLASMID DNA INTO THE HSV-1 GENOME 5511 waspurified byrepeated roundsoflimiting dilution and dot

blot analysis. The laxP insertion into the tk locus was

confirmedbySouthernblotanalysis(see Fig. 2) (30).Nytran filterscontainingtheelectrophoretically separated EcoRI or BamHIfragmentfromthe indicated DNAs were firstprobed withatk probe(2,416-bpEcoRI fragmentfrom pUCXI)(see

Fig. 3), strippedto remove the tk probe, and rehybridized

with aloxPprobe(350-bpNaeI-SmaI fragment frompBS64) (see Fig. 3). All probes used for dot blot and Southern analyseswerelabeledwith a Random Primed DNALabeling Kit(Boehringer Mannheim Biochemicals).

InvitroCre-mediated recombination. loxPshuttleplasmids

wererecombinedinto the KAT::loxlgenome in cell-freeCre reactions under conditions similar to those described by

Abremski andHoess(1)asmodifiedbySauer et al. (28). The DNAmixturewasincubatedat30°C for 30 to 40mininthe

presence of Cre buffer (50 mMTris-HCl

[pH

7.5], 33 mM

NaCl,10mMMgCl2)and 40 ngofpurified Creprotein (NEN Dupont Inc.,Wilmington, Del.). Forintermolecular recom-bination, reaction mixtures included KAT::laxl viral DNA

and a 20- to 50-fold excess of shuttle plasmid DNA, and

polyvinyl

alcoholwasaddedto afinal concentration of 1.7%

toenhanceintermolecular recombination (28,35). Recombi-nationreactionswereterminatedbyheatingsamples to 65°C

for 10 min, and reactionmixtures weretransfected into D6

cells to produce infectious virus. When recovery of the

shuttle plasmid from viral DNA was required, purified

KAT::108 viral DNA wasincubatedwithCrein theabsence ofpolyvinyl alcohol andtherecovered plasmidwas used to

transform DH5a by standardprotocols (27).

PurificationofCre-mediated recombinantviruses. Recom-binant viruses were identified on the basis of their

blue-plaque phenotype and purified by plaque purification and

limiting dilution. Transfection lysates were diluted and

platedatlowmultiplicity of infection (<0.01)ontoconfluent

D6 cell monolayers. Plaques weretested for lacZ activity with Bluo-gal

(GIBCO-BRL),

and viruses producing blue plaqueswerepickedas anagaroseplugand resuspended in

phosphate-buffered

saline (PBS). Individual plaque isolates

werethencloned bylimiting dilutionon D6cellsin

microti-terplates.Viruses wereconsidered pure when100% of the

plaques stained blue through three successive rounds of limiting dilution.

Assays for lacZ

activity.

Two assays wereused to detect lacZ activity in

developed

plaques.

For isolation ofviable virus, cultureswere rinsed withPBS

(10

mMsodium

phos-phate

[pH

7.1], 136 mM

NaCl),

overlaid with 300 mg of

Bluo-gal-0.5% low-melting-point

agarose

(GIBCO-BRL)

per

ml in MEM

supplemented

with10mM HEPES and5%fetal calf serum, and incubated at

37°C

until color

developed.

When isolation of viable virus was not

required,

infected cultureswerefixed andstained

by

ahistologicalmethod with

5-bromo-4-chloro-3-indolyl-,3-D-galactopyranoside

(X-Gal)

(Boehringer

Mannheim

Biochemicals).

Infected cultures wererinsedwith

PBS,

fixedfor 5 minatroomtemperaturein

2%

formaldehyde-0.3% glutaraldehyde

in

PBS,

and

incu-batedin a

chromophore

solution

containing

0.1%

X-Gal,

5

mM

K4Fe(CN)6

3H20,

5mM

K3Fe(CN)6,

and 2 mM

MgCl2

in PBS. Incubation continuedat

37°C

until color

developed

(usually after30to60

min),

and themonolayerswere

again

rinsedwith PBS.

RESULTS

Direct isolation of

Crelox-derived

recombinant viruses on

thebasis of

13-gal

reporter activity. The

general

strategyfor

K&T:oxi 03i -0

h.P,

1K

kb

-~~~~3~' 4.

bRa M13

wt

acMv-IaeZ~~~CI

1L:

1amP at gB HCMV-iac2 o*rPba MI3 kW

FIG. 1. HSV-basedCre-loxsite-specific recombination system. The target virus for site-specific recombination,

KAT::lox1,

was derived from KAT, and its genome contained (i) acis-acting site-specific recombination site laxP from bacteriophageP1(arrowhead) inserted into the tk coding sequence and (ii) a defective gB allele (AgB) due to a 969-bp

BstEII

(Bs) deletion in the gB coding sequence, including the transmembrane domain. In the example shown, thepJG108 shuttle plasmid was recombined into

KAT::laxl.

pJG108containedalaxP recombination site(arrow),an HCMVIEP-lacZ reporter gene cassette for identification of virus recombinants, and thewild-type gB allele from HSV-1 strain KOS. The polarities of both the viral and theplasmidlaxP sites are shown. pJG108 was inserted into the

KAT::loxl

genomeatthe tklaxPsite inacell-free Cre-mediated recombination reaction, and infectious virus was produced by transfection of the reaction mixture into D6 cells (8). The recombinant KAT::108 virus was diploid for the gB gene, containing both the deletedKAT::loxlallele

(AgB)

andthe wild-type KOS allele (wt gB). KAT::108 also yielded blue plaques in the presence of Bluo-gal or X-Gal because of the HCMVIEP-lacZ reporter gene.

using an HSV-1 shuttlevector systembased on the

bacte-riophage

P1

Cre-lox

site-specific

recombination

machinery

is shown in Fig. 1. The system consists of a

recipient

virus genome and ashuttle

plasmid,

each

containing

afunctional cis-acting laxPrecombination

site,

andtheCrerecombinase

protein.

In acell-free

reaction,

Cre-mediated

recombination

occurs

directly

between the twoloxP sites

(1)

such that the entire

plasmid

is inserted into the

recipient

viral genome.

Because the Crereaction is

reversible,

the shuttle

plasmid

can also berecovered from the viralgenome.

A recipient target

virus,

KAT::loxl,

was derived

by

markertransferofafunctionalloxP site into the tk locus of the gB- HSV-1 mutant KAT genome

(8).

Like

KAT,

the

KAT::loxl

recipientviruscontainsa

969-bp

BstEII deletion

withintheessentialgB gene andmustbe

propagated

onthe

gB-complementing

D6cell line

(8).

KAT::loxl

genomic

DNA was

analyzed

by

Southern

blot

(30)

toconfirmthepresence

of the

loxP

site within the tk

coding

sequence

(Fig. 2).

Shuttle

plasmids

containing

a functional loxP

site,

a

re

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[image:3.612.326.560.75.342.2]

J. VIROL.

bla bla

M13 oriP M13 oriP Kpnl

BamHl

: loxP BamHI

(BcllBamHt)

pJG108 pJG102

11.69kb 12.86kb

HCMV- 9B-1 HCMV- gB-1

_acZ (KOS321) /acZ (tsBS)

[image:4.612.64.295.78.432.2]

BaBamHamH lo Kpnl

FIG. 3. pJG108and pJG102 shuttle plasmids. Each plasmidwas derived frompBS+ and containsaloxP site-specific recombination site,anHCMVIEP-lacZreportergene,andanHSV-1gBgeneallele as thetestgene.ThegBgenes ofpJG108andpJG102arederived fromHSV-1 strains KOS andtsB5, respectively. Restriction sites usedtoclonetherespective fragmentsareindicated.

:- ..) to - a go

C

.1 .-: 'jr] E

K \\T

_P-.. 4;:

FIG. 2. Southernanalysisof KAT::loxlviral DNA. Pla

viral DNAs were digested as indicated, electrophorese

agarose, and transferred to a Nytran filter(Schleicher &

Keene, N.H.). The filter was sequentially hybridized i

labeledprobestotk(2,416-bpEcoRIfragmentfrompUCXI

the 1oxP-containing fragment (364-bp NaeI-SmaI fragm pBS64) (B). The tk probe was quantitatively removed hybridizationwith the loxPprobe. (C)Restrictionmapsof regions. Enzymes used were BamHI (B) and EcoRI positionsof the tkgene (arrow)and the tk SnaBI(Sn)sit

clone the 354-bp SmaI (S)-NaeI (N) frompBS64are als

Expected restrictionfragmentsizes in kilobasesarealsos

porter gene consisting of the bacterial lacZgene u:

transcriptional control of the strong HCMVIEP-e element, and one oftwo different alleles of the H'

genewereconstructed frompBSM13- (Fig. 3). The

gene was included for use as a marker for idt

recombinant virusescontaining the inserted shuttle I pJG108 contained the complete gB genefrom theM

HSV-1 strainKOS. pJG102 contained the HSV-1str mutantgBgene;themutantproteinproduct has bee tobedefectiveat 39°C andto induce polykaryocyt tion, or the syncytial (syn) phenotype, in infected

34°C (20).

The pJG102 and pJG108 shuttle plasmids were i

dently introduced into the KAT::Ioxl genome ina

Cre reaction. Infectious viruswasproduced bytran oftheCre reaction mixtures into the gB-complemei cell line. After 2 days, transfection lysateswere h.

andreplated on D6 cells andplaqueswere tested for P-gal

activity. Virus from individual blue plaques from each

ex-perimentwasisolated and subsequently clonedon D6cells by multiple roundsof limiting dilutions. Viruseswere

con-sidered pure when 100% of the isolates testedwere P-gal+

through three rounds of limiting dilutions. For each virus, '1''' insertion of the shuttle plasmidwasconfirmed bySouthern blot analysis (data not shown). Recombinant viruses were

namedby combiningthe KAT::prefixof the recipientvirus with the numeric suffix of the shuttle plasmid to yield KAT::102 andKAT::108, respectively.

__-__I Cre-mediated recombinationishighlyefficient. One

appeal-ing

property

of the

Cre-lox

system

for

introducing

DNA into the pseudorabies virus genome was the relatively high recombination efficiency achieved (28). Itwas therefore of l_ interest to determine the recombination efficiency of the HSV-basedsystem. Thepresenceof the lacZreportergene

on the inserted shuttle plasmid provided a convenient

smid and marker for

identifying

recombinant viruses

against

a

back-d in 1% ground of nonrecombinant viruses. SinceCrereaction

mix-Schuell, turesweretransfected into the

gB-complementing

D6

cells,

with 32p_ both the nonrecombinant gB- KAT::loxl and the

Cre-1)(A) and mediated gB+ recombinants should undergo productive

rep-ent from lication with essentially equal efficiencies. Therefore, the prior to percentage of P-gal+ plaques produced on D6 cells from a frelevant given transfectionlysate provided areliable estimate of the

(E). The recombinationefficiency ofaparticular recombination

reac-eused to tion mixture.

shown.

DNA from fourindependent preparationsof

pJG108

was used in separate Cre reactions witha single preparation of

KAT::loxl viral DNA, and viruses in the resulting transfec-tion lysates were examined for frequencies of recombina-nderthe tion. The resultsarereportedasthe number of blue plaques

SV

gB-n

per total number of plaques (Table 1). The Cre-mediated reporter

entifying

plasmid. TABLE 1. Cre-mediated recombination efficiency vild-type

rain tsB5

nshown e forma-lcells at indepen-cell-free isfection

nting D6 arvested

Plasmid' Dilution' No.of blue No. oftotal % blue

plaques plaques

plaquesc

None -7 0 120 0

pJG108 -5 8 40 20

pJG108 -5 5 36 14

pJG108 -4 26 233 11

pJG108 -5 5 41 12

a Cre reactions containedeitherno shuttle plasmid DNA or DNA from one

of fourpJG108 plasmidDNA stocks.

bDilution oftransfected cell lysate used for blue-plaque assay.

cPercentblue-plaquevalues are (blue plaques/total plaques) x 100.

5512 GAGE ET AL.

A

B

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[image:5.612.343.535.72.333.2]

INSERTION OF SHUTTLE PLASMID DNA INTO THE HSV-1 GENOME TABLE 2. Rescue of gB- phenotype ofKAT::1oxl by

Cre-mediated insertion ofpJG108 orpJG102

Titer(PFU/ml) D/eo Plaque

Virus Titer_(PFIJ/ml)_ D6/Vero morphology

efficiency

D6 Vero D6 Vero

KAT::laxl 1.8 x 108 0 0 Syn+ NAb

KAT::108 8.5 x 108 9.1 x 108 1.07 Syn+ Syn+ KAT::102 1.7 x 107 1.8 x 107 1.06 Syn Syn

a For each virus, efficiencyis defined as thetiteron Verocells/thetiter on

D6 cells.

bNA,notapplicable.

recombination efficiencies ranged from 11 to 20% and aver-aged about 15%.

Complementation of the gB-

phenotype

of KAT::Ioxl. Experimentswere carried out to confirm that expression of

thepolypeptidefromthe shuttle plasmid would complement the defective gB gene at its nativeviral locus and that the

phenotype ofthe resulting recombinant virus would reflect thegenotype of the plasmid copy of the gene in thetklocus.

KAT::108and KAT::102 both encode functional gB

polypep-tides on their respective shuttle plasmids, with KAT::108

encodingthewild-type gB polypeptide and KAT::102

encod-ing themutant gB polypeptide from HSV-1 strain tsB5. The

tsB5 gB polypeptide includes a ts mutation within the

external domain (10) and a syn mutation (Arg to His) at

residue 858within the cytoplasmic domain (5, 6, 10).

There-fore, KAT::108 andKAT::102 were used to demonstrate (i)

that complementation of the gB- defect of the parental

KAT::loxl virus after insertion of the pJG108 or pJG102 shuttle plasmid occurred and (ii) that the tsB5 mutant gB

polypeptide encoded by pJG102 conferred both mutant

phenotypes (temperature sensitivity and syncytial plaque

morphology)onKAT::102.

In an initial experiment, both the KAT::108 and the

KAT: :102 viruseswereshowntoreplicate normallyon Vero

cells inplaqueassays (datanotshown). Therefore, titersof

KAT::108 and KAT::102 stocks preparedonVerocells anda

KAT:

:loxl

stockgrown on D6cellsweredeterminedonboth

D6andVerocells.Asexpected,

KAT::lQxl

developedatiter

onthegB-complementingD6cell line butnot onVerocells (Table 2). In contrast, KAT::108 and KAT::102 each grew

equally well on either cell line.The plaque

morphology

of each virus was also examined. KAT::102

produced

the mutantsynplaque

morphology

onboth cell lines

(Table 2).

Therefore,

expression

offunctional gB

polypeptides

from the inserted shuttle

plasmids complemented

the deletion within thegBgeneatits native virallocus and

expression

of

the allele from

pJG102

conferred the syn

phenotype

on KAT::102. The KAT::102recombinant viruses also

demon-strated temperature-dependent virus

growth.

The ratio of KAT::102 recombinant virus

produced

at 39°C

compared

with that

produced

at34°Cwas

approximately

5 x

10-4,

an indexof temperature

sensitivity

similartothat of theparent virustsB5.KAT::108grewequallywellatboth 34 and

39GC.

Efficient Cre-mediated recovery of intact shuttle

plasmids

from KAT::108 viral DNA. Sauer et al.

(28)

demonstrated thatinserted shuttle

plasmid

DNAcouldbeexcised fromthe

pseudorabiesvirus genome in a

Cre-dependent

mannerbut

did notexamine the

integrity

of the recovered shuttle

plas-mid. If the

Cre-lax

methodology

is to be used as a true shuttlevector system, the inserted shuttle

plasmids

recov-ered from the viral genome must be intact. The shuttle

a0

Miniprep DNAs

2

3

4

5

6 7 8

9 10

FIG. 4. Cre-mediated rescue of intact shuttle plasmid DNA from KAT::108 viral DNA. KAT::108 viral DNA was reacted with Cre recombinase and transformed intoDH5a to propagate the rescued shuttleplasmid. Miniprep DNAs were isolated from 10 independent colonies, restricted withPstI, andelectrophoresed in 1% agarose. All10miniprep DNAs are identical to the pJG108 control.

plasmid was therefore recovered from extracted KAT::108 viralDNA and compared with the original pJG108plasmid stock by restriction enzyme analysis. The plasmid was

released frompurified KAT::108DNAin anintramolecular Crereaction andpropagated after transformation ofDH5a. Small batches of

plasmid

DNA(17) were prepared from 10

isolated colonies, and their PstI restriction patterns were

compared with that of pJG108. All 10 miniprep DNAs

showed restrictionfragment

patterns

that were identical to

that of

pJG108

(Fig. 4), demonstrating

that large shuttle

plasmids couldbe recovered intact from the viral genomeby Cre-mediated recombination.

DISCUSSION

In this report, we describe the

adaptation

of the Cre-lax site-specific recombination system of bacteriophage P1 for usewith HSV-1 and

investigate

the

potential

usefulness of

this

approach

for the

genetic

analysis

of an essential viral

gene.

Site-specific

recombination mediated

by

the Cre

re-combinase

protein

occurredbetweentwoloxP

sites,

oneon the shuttle

plasmid

and the other on the

recipient

virus genome. A

constitutively

expressed

lacZ reporter gene cassette on the shuttle

plasmids

allowedeasy identification of recombinant viruses on the basis of their

blue-plaque

phenotype. Because Cre-mediated recombination was

re-versible,the

plasmids containing

cloned

copies

of the essen-tialgBgene could berecoveredfor further

analysis by

using

a second Cre reaction. Recovered shuttle

plasmids

had a

restriction fragment

pattern

identical to that of the

original

plasmid.

Expression

offunctional

gB

polypeptides

from the inserted shuttle

plasmids

at the tk locus

complemented

a

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[image:5.612.60.305.94.176.2]

5514 GAGE ET AL.

lethal defect in thegBgene. Expression of the tsB5gBallele conferred the ts and syn phenotypes on the recombinant

virus,

demonstratingthat thisapproach is useful for

testing

mutantconstructs foraltered phenotypes.

TheCre-loxshuttlesystem offers anumber of

advantages

fordetailed

genetic

analysescompared with a strategy based on markertransfer. (i) Since recombination occurs

through

the

34-bp

cis-acting loxP site, no homologous viral

flanking

sequencesarerequiredtofacilitaterecombination,

minimiz-ing clonminimiz-ing

steps that may be needed for marker transfer. Moreover, this site-specificrecombinant event avoids

poten-tial

problems

related to rearrangement of the

integrated

DNA which sometimes occurs in marker transfer

experi-ments.This isparticularlyimportant in experiments

involv-ing

the insertion of large DNA fragments. Although the

Cre-lox approach

does introduce plasmid DNA sequences

along

with the testgene(s),thefunction of either the reporter or the gB gene was not affected by the presence of these nonviral sequences. (ii) Therecombination frequencies

ob-served for the HSVCre-lox system were even higher than those

reported

forthepseudorabiesvirus-based system and areasmuchas 1 order ofmagnitude (10to 20% versus<2%)

greater than those typically observed in marker transfer

experiments.

The constructs containedboth a complete test gene and a reporter gene such that recombinants can be

readily

detected and

purified

on the basis of reporter gene

expression.

Also,inthese studies, theexpected

complemen-tation of the deleted gB allele by the plasmid-encoded

wild-type

and tsB5 gB proteins could have been used as a means ofselecting for recombinants by transfection ofthe

Cre-mediated

recombination reaction mixtures directly into

noncomplementing

Vero cells. (iii) Unlike DNA introduced

by

markertransfer,genesintroducedonshuttle plasmids are

directly

recoverable from theviralgenomein a cell-free Cre reaction without additional cloningsteps and are thus readily

available for further analysis. Thisis particularly important for studies requiring the generation and testing of a large

panel

of mutants. For example, randomly mutagenized genescanbeintroduced intotheviral genome and tested for a

particular

phenotypesuch assyncytialplaque morphology, and then only those mutant constructs demonstrating the

desired

phenotype

may be recovered from plaque-purified viral DNA for analysis by DNA sequencing. Thus, the

ability

to easily recover inserted shuttle plasmids from the viralgenomeshouldmake a priordetailed analysisof genetic constructs unnecessary. (iv) Finally, the

Cre-lox-based

re-combination system should be useful for viral systems for

which marker transfer procedures are currently difficultor

unavailable. These includethecytomegaloviruses, Epstein-Barrvirus, andvaricella-zoster virus.

ACKNOWLEDGMENTS

Wethank Neal DeLuca,William Goins, RameshRamakrishnan, and Stan Person forsuggestions,discussions of the data, andcritical readingof themanuscript.

Thiswork was supportedby U.S. Public Health Service grants GM34534, A126937, and A118228 from the National Institutes of Health.

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