Improved cell survival by the reduction of immediate-early gene expression in replication-defective mutants of herpes simplex virus type 1 but not by mutation of the virion host shutoff function.

JOURNALOFVIROLOGY,Oct. 1994,p.6347-6362 0022-538X/94/$04.00+0

Copyright (C 1994, American Society for Microbiology

Improved Cell Survival

by the Reduction

of

Immediate-Early

Gene

Expression in Replication-Defective

Mutants

of

Herpes

Simplex Virus Type 1 but

Not

by Mutation of

the

Virion Host Shutoff

Function

PAULA. JOHNSON,* MING JINGWANG,ANDTHEODORE FRIEDMANN

Department of Pediatrics, CenterforMolecular Genetics, University ofCalifomia,

SanDiego, LaJolla, California 92093-0634

Received 4 March 1994/Accepted6 July 1994

Derivatives of herpes simplex virus type 1 (HSV-1) have elicited considerable interest as gene transfer

vectors because of theirability to infect a wide range of cell types efficiently, including fullydifferentiated

neurons. However, it has been found that infection of many types of cell with vectors derived from

replication-defectivemutantsof HSV-1 isassociated with cytopathic effects (CPE).We have previously shown

thatviralgeneexpression playedanimportant role intheinductionof CPE caused byanHSV-1mutantdeleted

for theessential immediate-early gene3 (IE 3) (P. A. Johnson,A. Miyanohara, F. Levine, T. Cahill, and T.

Friedmann,J.Virol. 66:2952-2965,1992). We have investigated which viralgenesmight be responsible for CPE

by comparing the ability of each of the individual genes expressed by an IE 3 deletion mutant during a

nonproductive infectiontoinhibit biochemical transformation after cotransfectionof BHKorCV-1 cells with

aselectablemarkergene.Transfection of IEgenes 1, 2, and 4individually allcaused amarkedinhibition of

colonyformation,whiletransfection ofIE 5 and the large subunit of ribonucleotide reductase had little effect.

These results suggested that itwould benecessaryto mutateorreduce the expression of nearly allHSV-1 IE

genestoreducevirus-induced CPE. Therefore,wehave used VP16mutants,whichareunabletotransinduce

IE gene expression (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol.

63:2260-2269, 1989),toderivetworeplication-defective strains: 14HA3,which is deleted for both copies of IE

3,andin1850A42,whichhasadeletionintheessential earlygeneUL42. The IE 3-VP16 doublemutant,14HA3,

is significantly less toxic than a single IE 3 deletion mutant over a range of multiplicities of infection, as

measured in a cell-killing assay, and has an enhanced ability to persist in infected cells in a biologically

retrievable form.Incontrast, theUL42-VP16doublemutant,in1850A42, showed reduced toxicity onlyatlow

multiplicities of infection. To test the roleofthevirion host shutofffunction as anadditional candidateto

influencevirus-inducedCPE,wehave introducedalarge insertion mutation intothevirionhost shutoffgene

ofanIE 3 deletionmutantandthe doublemutant14HA3. Mutation of thisgenedidnotreduce thecytotoxicity of eitherstrain. Theseresults demonstrate thatlong-term survival of cellsinfected withreplication-defective HSV-1 mutantscanbeenhancedthrough genetic manipulations that reduce viralgeneexpression.

Approaches to human gene therapy take advantageof the

highefficiencyof viralvectorsinintroducing foreigngenesinto

mammalian cells (56).To be useful inmostcases, theforeign

genes should be expressed stably and efficiently, and the

processofgenetransfer shouldnotproducecelldamage.Some vectors,suchasthose derivedfromretroviruses, satisfymostof theserequirements,butmostretroviralvectorsarenotcapable ofgenetransfertopostmitoticcells. Therearemanyinstances inwhichgenetransfertonondividingcells would bedesirable, whethertopermitstudies ofgenetic manipulationin differen-tiated cell types or for more applied therapeutic purposes.

Herpes simplexvirus type 1 (HSV-1) has attracted

consider-able interest for itspotentialas agenetransfer vector. Much is known about HSV-1atthe molecularbiological level,it is able to infectawide rangeofreplicatingand nonreplicating cells, and it can maintain a lifelong latent infection in sensory

neurons(for recentreviews onHSV-1vectors,see references 5 and35).

*Correspondingauthor.Mailingaddress:Dept.ofPediatrics,

Cen-terfor MolecularGenetics, UniversityofCalifornia at SanDiego,La Jolla, CA 92093-0634. Phone: (619) 534-1419. Fax: (619) 534-1422. Electronic mail address: [email protected].

Vectors derived fromreplication-defectivemutantsor atten-uated strains of HSV-1 haveprovedsuccessful for thetransfer

andexpressionofforeigngenesinthe mammalian braininvivo

(8, 11, 17) and in primarycentralnervous system neurons in

vitro(30). Thetargetcells for infection with HSV-1vectorsare

not limited to neurons, as demonstrated by the high but

transient levels of circulating transgene products in mice

following injectionof the liver with HSV-1 vectorsexpressing

canine factor IX or hepatitis B virus surface antigen (43).

However,inmostcases,attempts toachievelong-term

expres-sion offoreigngenes, at least in cellsotherthan those of the

peripheral nervous system in vivo, have been hampered by

cytotoxicity and/or shutoff oftransgeneexpression.

We have studied the cytopathic effects (CPE) caused by infection withvectors derivedfrom an HSV-1 mutant called

D30EBA (49),deletedfornearlythe entirecodingportionof

immediate-early gene 3 (IE 3), whose product, Vmwl75

(ICP4), is the major transcriptional activating protein of

HSV-1(51).Since Vmwl75iscritically requiredforexpression of viralearlyand lategenes,anIE3mutantexpressesveryfew viral genes and iscompletely replication defective (9, 24, 49,

51). However,wehave demonstratedsignificant CPE,

includ-ingfragmentationof cellularDNA andcytoplasmicblebbing,1

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to 3 days after infection with an IE 3 mutant (29). We also

found thatwaspossible toreduceCPEbytwo methodswhich reduced viralgeneexpression: UVirradiation of thevirusand

pretreatment of cells with interferon (29). These results

sug-gest that one or more of thefew viral genesexpressed in the absence ofVmwl75were, at least in part, responsible for the CPE.

The viral genes efficiently expressed by an IE 3 deletion mutant include IE 1, whose product, VmwllO (ICPO), is a

general transactivator of transcription but is not absolutely

essentialfor virus replication (6, 12, 65);IE2,whoseproduct,

Vmw63 (ICP27),isrequired forexpression of truelategenes, full levelsofviral DNAsynthesis,andmodulationofearlygene

expression and which, in transfection experiments, can act as

an activatororrepressor oftranscription incombination with Vmwl75 and Vmwl10 (38, 55, 60) (Vmw63has recentlybeen

shown to affect mRNA splicing and stimulate viral mRNA 3'-end processing [42, 58]); IE 4, whose product, Vmw68

(ICP22), isrequired forefficientvirus replication insome cell

types(59); IE5,whose product,Vmwl2(ICP47), hasrecently

been shown to inhibit antigen presentation to CD8+ T

lym-phocytes in infected human fibroblasts (68); and the gene

encoding ICP6, which is the large subunit of ribonucleotide

reductase,whichis notessential forvirusreplication intissue

culture (20). Wehave previously tested the cytopathogenicity

ofHSV-1 mutantsbearing lesions in one or more of thefive individual IEgenesanddetermined that noneofthe IEgene

products alone areresponsibleforCPE (29). In addition, not

everycellinfectedwiththe IE3mutant issubjecttocelldeath

(29),afindingconsistentwith thenotionthatathresholdlevel

ofthe IEgene productsmay berequiredfortheinductionof

CPE and that cell survival may be enhanced by reducing or

turning offviral geneexpression.

The first part of the present study was aimed at the identificationof the HSV-1 IEgenesresponsiblefor inducing

CPE during infection with an IE 3 mutant. As an assay to

measure the potential for CPE, we have cotransfected cells

with the bacterial neomycin phosphotransferase gene (neo), which confers resistance to the neomycin analog G418, as a

selectable marker, together with HSV-1 sequences that may

interfere with stable biochemical transformation. We postu-latedthatfewerneo-transformed cells would beobtained from

cotransfections which include a gene encoding a

cytopatho-genic product. As a control for promoter competition or for

the presence of spurious inhibitory sequences, we have

in-cludedplasmids bearingmutationswithinthecoding regions of

theHSV-1 genesbeingtested. Ourresultsindicatethat IE 1,

IE 2, and IE 4 all encodecytopathogenic geneproducts. We havealsofoundthatIE3causesinhibitionoftransformation in

CV-1 cells butnot inBHKcells.The ICP6geneandthe IE5 gene didnotdemonstrateinhibition oftransformationin this assaysystem.

Animportant regulatorofHSV-1 IEgeneexpression isthe

virion polypeptide VP16 (Vmw65), which functions to

stimu-lateIEgenetranscription atthe earlieststagesof infection (3,

7) through formation of a complex containing VP16 and

cellular transcription factors, including Oct-1, which together

specifically bind the IE promoter element TAATGARAT,

whereRisapurine (45,52,66).Aceetal. (2) constructedan

HSV-1mutant, in1814,whichhasa12-bpinsertionin theVP16 gene which abolishes the ability of VP16 to transinduce IE

gene expression. However, this virus is stillable toreplicate,

particularly at high multiplicities of infection (MOIs). These

resultssuggestthat itmightbepossibletogenerateavirus that

does not induce CPE during a nonproductive infection ifthe

VP16 mutantin1814were furthermutated to render it

repli-cation defective. For that reason, we haveconstructed viruses

with deletions in either IE 3 or UL42 aswell asthe insertion

mutation in

VP16,

designated

14HA3 and

in1850A42,

respec-tively. In

comparison

to their VP16-intact counterparts, we

have found that 14HA3 has reduced

cytotoxicity

over a range

of

MOIs,

whereas in1850A42 is less toxic

only

atlow MOIs.

The virion host shutoff(vhs)function is acomponent of the

virion encoded bygeneUL41

(40),

which has been

thought

to

contribute to the

cytotoxicity

of HSV-1 vectors. The vhs

function suppresses translation of

preexisting

mRNAs and

increases the turnover of both viral and cellular nascent

mRNA

(33, 47,

53), although

the

strength

of this function may

varybetweenstrains and is weak in HSV-1 strain17+

(15).

The

vhsmutantvhs-1 isreplication competent (53), althoughinan

earlier

study,

it did not show reduced

cytotoxicity

compared

with itsparentalwild-type strain

(KOS)

evenwhen viralDNA

replication was inhibited

(29).

In the present

study,

we have

tested morerigorouslytheinvolvement of vhs in the induction

of CPE by replication-defective mutants of strain

17+

by

introducing alarge insertion mutation into the vhs gene ofan

IE 3 deletion mutant and alsointo the vhs gene of the

newly

constructed IE 3-VP16double mutant 14HA3.

Analysis

ofthe

resultingmutants indicated that vhs doesnotcontributetothe

cytotoxicity inducedbythis strain of HSV-1 duringa

nonpro-ductive infection. To determine whether a potential

involve-ment of vhs in virus-induced cytotoxicity was obscured by the

effectsof viral gene expression, we measured cellviability after

infection with UV-irradiatedvhsmutants of strains KOS and

17+ and their parental virus strains, but again, there was no

significant difference in cytotoxicitywhich correlated with the

vhsmutation.

MATERLILSANDMETHODS

Cells and viruses. All cell lines were propagated in

Dulbec-co'smodificationofEagle'sminimal essential medium

(DME)

containing 10% fetal calf serum. BHK TK- and CV-1 cells

were used to assay the inhibition of colony transformation.

TO-119 primary human diploid fibroblasts were used to assay

cellkilling and the ability of virus to persist in infected cells.

Cells used to support growth of IE 3 deletion mutants were

RR1(29) and E5 (obtained from N. A. DeLuca, University of

Pittsburgh,Penn.) (10);V9 cells were used to supportgrowth

of UL42 deletion mutants (27). RR1, E5, and V9 cellswere

passaged in medium containing 400 ,ug of G418 (geneticin;

GIBCO BRL, Grand Island, N.Y.) per ml.

All the viruses used in this study were originally derived

from HSV-1 strain 17+ except where stated below. The VP16

mutants in 1814 and in1850 were generously provided by C. M.

Preston (Medical Research Council Virology Unit, Glasgow,

Scotland). The mutant in1850 was derived from in1814(2) by

insertion of theEscherichia coli lacZ gene into the thymidine

kinase (TK)coding sequence(Sla). The IE 3 deletion mutant

D30EBA was constructed by Paterson and Everett (49). CgalA42 contains a deletion in the UL42 coding sequence (27).

Viruses containing the in1814-specific lesion in the VP16 gene

wereprepared and titrated by infecting cells in the presence of

5 mM hexamethylene bisacetamide (HMBA; Sigma) for the

first 24 h, as described before (39). This step significantly improves both the titer and the particle/PFU ratio of VP16 mutants. An ICP8 deletion mutant derived from strain KOS, d301 (19), was used in the superinfection rescue procedure and

was kindly provided by D. Knipe (Harvard Medical School,

Boston, Mass.). The virion host shutoff mutant vhsl (53) and wild-type strain KOS were kindly provided by G. S. Read (University of Missouri-Kansas City), and the IE 3 deletion

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CYTOTOXICITY OF REPLICATION-DEFECTIVE HSV-1 MUTANTS 6349

mutantD30EBA was kindly supplied by R. D. Everett

(Med-ical Research Council Virology Unit).

Plasmids. The plasmids used in the colony inhibition assay

areshown in Fig. 1. The IE 1 gene contained in plasmidplll,

theIE 1 deletion mutantpllOdel4,the IE 3 gene inpl75,and

two deletion mutants of IE 3, pD8 and pD9, were obtained

from R. D. Everett(48,50). PlasmidpKl-2 contains IE 3 under

the control of itsown promoterand was obtained from N. A.

DeLuca (10). The IE 2 gene is contained in a 2,448-bp

BamHI-HpaIfragment in plasmid pIE63 (29), from which the following derivatives were made: pIE63+ contains an

addi-tional 815-bpHincII-BamHI fragment adjoining the 5' end of

IE2; pXin63+ contains anXhoIlinker inserted at the SalI site

ofpIE63+ (disruptionof the IE 2 sequenceatthisSallsite has

been shownto impairthe activator and repressor functions of

Vmw63 in a transient assay system [22, 55]); and pX63.1

containsanXhoIlinker inserted atthe site ofa28-bpdeletion

betweentwoRsrIIsites inpIE63.The IE 4 gene is contained in

plasmid pIE68, and the derivation ofpIE68A,whichcontainsa

425-bp deletion between the SstI andHindlIl sites of pIE68

andaninsertionof 176 bpofMoloneymurine leukemia virus

DNA, has been described(29).PlasmidpZ4containsa4.6-kb

human cytomegalovirus (HCMV)-lacZ fragment inserted

be-tween the SstI andHindIIIsites ofpIE68, asshown in Fig. 1.

The IE 5 gene is contained in pIE12 as an EcoRI-BamHI

subfragmentof BamHI-x. This fragmentwasinserted between the HindlIl and BamHI sites of pl,

replacing

the

firefly

luciferase gene (luc) (28), so that IE 5 is terminated

by

the

simian virus 40

(SV40) polyadenylation signal

present in pl; pIE12doesnotcontaincompleteopen

reading

frames for the overlapping3' coterminal genesUSl0andUS11

(41).

Plasmid

pZ5 was derived from BamHI-x as described before

(29).

Plasmids pKHF, which contains the large subunit of

ribonu-cleotide reductase (ICP6), and

pKHFA,

which contains a

deletion within the ICP6

coding region,

were obtained from

S. K. Weller (Universityof

Connecticut)

(20).

All the HSV-1

genes described above were derived from strain

17+

except

those onplasmids

pKl-2,

pKHF, and

pKHFA.

The plasmids used to confer resistance to G418 were

pSV2neo

(62)

and pHneo

(28),

which contain the SV40 enhancer-promoter and the human

hypoxanthine

phosphori-bosyltransferase

(HPRT)

gene promoter,

respectively,

driving

neo.

Theplasmidusedto delete IE 3

coding

sequencesfromthe

viral genomewaspIllH.Thiswasderived from

pdelI11

(49)

by

insertion of a

587-bp

HaeIII

fragment

from

pUC19

into the

EcoRI site,

marking

the IE 3 deletion

by

blunt-end

ligation

(see Fig.

3).

The

plasmid

used to

disrupt

the

coding

sequence

of the vhs genewas

pUL41-lacZ.

This

plasmid

containsthe5'

portionof the vhs gene from

pBam-i-Sst,

into theNrulI siteof

which was

inserted, by

blunt-end

ligation,

a 4.6-kb BamHI

fragment

containing

an HCMV-lacZ cassette from

pON249

(63) (see

Fig.

3b).

Other

plasmids

used in this

study

were

pBaml,

which

contains the HSV-1

BamHI-l

fragment

in

pUC19,

and

pUL42A,

which contains a

1,258-bp

deletion of the UL42

coding

sequencewhichis marked

by

an insertion ofa

280-bp

PstI-PvuII fragment from

pBluescript

KSII

(27). pMC1

con-tainsthe

coding

sequenceof VP16andwas

kindly

provided

by

C. M. Preston

(7).

Transfections and

colony

inhibition assay. We have used

thecalcium

phosphate

method of Graham andvander Eb

(21)

for all transfections. For the

colony

inhibition assay,

freshly

trypsinized cells were seeded on six-well cluster dishes at a densityof

105

cells per 35-mm well 1

day

prior

totransfection.

The cells were cotransfected in

triplicate

with 0.25 ,ug of

UL us

a) - | IRL I IA ITRS

UL5f].f:[UL55 (Fjt¶- (E:Z1J JED)CUS2 (.&L

IE2 IEl IE3 IE4 IE5

b) _pIE63 BSaHp HcB

pIE63+ i Hc Xh pXin63+ r

B Sa

pX63.1 6

(AsRs)

Xh

HpSa Xh S R SH Ml

pp 4plp111 I- --- plE68 1Pp~~~~~~~~~~6pl11Odel4 p Z4

lacZHCMV

pKl-21

-S B

p175 i

-= SV40

Dg DB En.Pr

c)

Vmwl75

4

N~~~~~~~~~~~~~~~~~~~~~~~~~~~

_.

VmwllO0

B,SApR

I plEl12

IacZ HCMV

K\G) N.(3b c4.' S

9 .99.

(N

.P9

c.1

9\ V.l.

9 9

Vmw63 _

BHK

cv-1

FIG. 1. Location ofthe HSV-1 IEgenesin the viral genome and the structures of IE

gene-containing

plasmids. (a)

The

right-hand

portion

oftheviral genome isshown,

including

partof the

long

unique

region

(UL),

the internal

long

repeat

(IRL),

and the short

unique

region

(Us)

bounded

by

internaland terminal shortrepeats

(IRS

and

TRS,

respectively).

The IE

transcripts (arrows)

and their

coding

regions (shaded

boxes)

andadditional open

reading

frames thatmap closetoIE2

(UL53

and

UL55)

andIE 4

(US2)

are

depicted

belowthe genomestructure.Notethat

copies

ofIE 1and3arepresentineach repeat

(not shown). (b)

Structuresofthe IE

gene-containing plasmids

and their mutant derivatives. The

origins

and derivations of these

plasmids

aredescribedinMaterials and Methods. D8 andD9referto

deletions within

p175,

contained in

plasmids pD8

and

pD9,

respec-tively.

Ap, ApaLI;

B, BamHI; H,

HindIII;

Hc, HincII;

Hp, HpaI;

P, PstI; R, EcoRI; Rs, RsrII;S,SstI; Sa,

Sall;

Xh,

XhoI;

En/Pr,

enhancer-promoter.

(c)

Immunoblot detection of the

products

ofHSV-1 IE genes1,2,and 3.BHKand CV-1 cellsweretransfected with

plasmids

carrying

theindividualIEgenesasindicated.After48h,thecellswere collected

by

scraping,

andtotal-cell

lysates

were

prepared

by boiling

in sodium

dodecyl

sulfate

(SDS) gel loading

buffer

(36).

Fractionsof the cell extracts were run on

SDS-polyacrylamide gel electrophoresis

(PAGE),

andthe

proteins

were electrotransferredontonitrocellulose membranes. The IE gene

products

were detected

by

monoclonal antibodies

against

HSV-1 Vmwl75, VmwllO,andVmw63,obtained fromAdvanced

Biotechnology,

withthe enhancedchemiluminescence kit fromAmersham.

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[image:3.612.316.550.95.470.2]

pSV2neo

as aselectable markerto confer resistance to

gene-ticin and 2.5 ,ug of test

plasmid

DNA

containing

HSV-1

sequences. Sixteen hours after

transfection,

the medium was

replaced,

and afterafurther 24 h of

incubation,

the cellswere

split by

dilution factors of 1:4 and 1:50 and seeded onto

100-mm

plates

in selection medium

containing

G418

(400

,ug/ml).

Colonieswere counted 10to 14

days

later.

Generationof recombinant viruses.

(i)

Isolation of

D30HA3,

14HA3,

and14HRA3. RR1 cells

(2

x

105)

werecotransfected

with

(i)

2,ug ofa1:1mixture ofintact and

BamHI-SstI-cleaved

pIllH

and

(ii)

2 jig of

D30EBA

or in1814

genomic

DNA.

After24

h,

the cotransfection mixturewas

removed,

and fresh

medium

containing

5 mM HMBA was added to the cells to

stimulate HSV-1 IE gene

expression

intheabsence of

transin-duction

by

VP16

(39).

The cells were

replenished

with fresh

medium

(without HMBA)

after an additional 24 h. After

severalmore

days

of incubationat

37°C,

whendiscreteareasof

CPE had

formed,

the cellswereharvested and

freeze-thawed,

and virus progeny in the

supernatant

were

plated

out at

appropriate

dilutionsonto10-cmdishes of

E5

cell

monolayers

inthe presence of 5 mM HMBA. Thenext

day,

thecellswere

overlaidwithmedium

containing

0.8%agaroseinthe absence

ofHMBAandincubated foranadditional 3to4

days

toallow

theformationof

plaques.

Recombinant virus which had

incor-porated

the IE 3 deletion mutation contained in

pIllH

(marked by

the

pUC19

HaeIIIDNA

fragment)

were detected

andisolated

by

several rounds of in situ

plaque

hybridization

as

described

previously

(27), using

the

587-bp

HaeIII

fragment

from

pUC19

as a

probe,

radiolabeled

by

the method of

Feinberg

and

Vogelstein (14).

The

genomic

structure of

vi-ruses was determined

by

Southernblot

analysis

of total DNA

prepared

from 16-mm-well cell cultures infected with

plaque

isolates,

picked

after each round of

plaque

hybridization.

In this manner, the

incorporation

ofthe

pUC19

DNA

fragment

into the correct location of both short repeats of the HSV-1

genomein

place

ofIE3 sequenceswasmonitoredtoallow the

isolation and

purification

of recombinants derived from

D30EBA

and

in1814,

which were

designated

D30HA3

and

14HA3,

respectively.

Markerrescueof the mutated VP16 gene

in 14HA3was

performed

by

cotransfection of RR1 cells with

14HA3

genomic

DNAand

pMC1,

which contains the

wild-type

VP16 gene.

Larger

plaques

which grew in the absence of

HMBAwere

picked

and

purified,

andtheir

genomic

structure

was

analyzed

asdescribedabove for loss of the BamHI linker

inthe VP16 gene. Three suchisolatesallexhibited thecorrect

BamHI

restriction

profile,

anda

large

preparation

made from

the firstofthesewas

designated

14HRA3.

(ii)

Isolation of

inl850A42.

BHK TK- cells

(106)

were

co-transfected with 10

pLg

ofin1850DNA and 10 ,ug

(total)

ofan

equal

mixture of intact and BamHI-cleaved

pUL42A

DNA

(27).

Progeny

viruseswere

plated

ontoV9 cell

monolayers,

and the

resulting plaques

were screened for the presence of the

280-bp fragment

of

plasmid

vectorDNA

marking

thedeletion

ofthe UL42 gene, as described above. Stockswere prepared

from a

plaque-purified

isolatewhich could

only

form

plaques

on V9 cells andwhich exhibited thecorrectrestriction digest

profiles corresponding

to the UL42 gene deletion and the

VP16 gene insertion mutation

(26).

This virus was named

inl850A42.

(iii)

IsolationofA3vhsZand 14HA3vhsZ.

Disruption

of the vhs

coding

region

of

D30EBA

and 14HA3 was achieved by cotransfectionof RR1 cells with

pUIA1-lacZ

DNA,

linearized

by digestion

with

XhoI

and the

appropriate

viral genomic DNA. Recombinantvirus in the

resulting

progeny were

de-tected

by

the appearance of blue

plaques

after

plating

on

monolayers

of

E5

cells and

staining

with X-Gal

(5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside) as

previously

de-scribed (29). The genomicstructuresof virus isolates

carrying

the intended insertion mutation in the vhs genes ofD30EBA

and 14HA3 were confirmed

by

Southern blot

analysis,

and

these isolates were designated A3vhsZ and

14HA3vhsZ,

re-spectively.

Yield of progeny virus.E5orV9 cells

(as

appropriate)

were

seededat adensityof 3 x

105

cellsper 35-mm dish. Afteran

assumeddoubling

by

24h,the cellswereinfected with virusat

an MOI of 1 or 0.01 PFU per cell. Unabsorbed virus was

removed by washing 1 h

later,

and the

monolayers

were

incubated foranadditional 19 hat37°C.Infected cultureswere

maintained continuously for the 24-h

period

in either the

presence or absence of 5 mM HMBA. The cellswere then

scraped into the medium anddisrupted

by freeze-thawing,

and

virus in theresultingsupernatantswastitrated

by plaque

assay

onpermissive cells in the presence of 5 mM HBMA and 10%

pooledhuman serum.

Cell

viability

assays. The effect of virus infection on cell

survival and growth,as measuredby thenumber of adherent

cells at 3 days postinfection, was determined as follows.

Human fibroblastswereseededat adensityof 5x 104cells per

16-mm well. The cellswereinfected in

triplicate

16 hlaterwith

virusatMOIs of0.1, 0.25,0.5, 1.0, 5.0,7.5,10,and 25 PFU per

cell. Aftera1-habsorptionperiod, the cellswerewashed and

replenished with fresh medium. At 3

days postinfection,

loose

cells and debris were removed

by washing

with

phosphate-buffered saline(PBS), and the adherent cellswereharvested

by

trypsinization and counted witha Coultercounter.The titers

of the virus stocks used in this cell viabilityexperiment were

also determined intermsofinfectious genomeunits(IGU)per

ml, as follows. Human fibroblasts (106) were infected with

either a standard volume of virus stock(25

,ul)

or a constant

MOI (e.g., 5 PFU per cell). The cellswerewashedthreetimes

aftera 1-habsorptionperiodand thenincubated for 1 hmore

at37°C. The cellswerethenharvested inPBS,and cell nuclei

were isolated from outer cell membranes and

cytoplasmic

components following Nonidet P-40lysis. Nuclear DNAwas

prepared, and 2.5-,ug aliquots were digested with BamHI

alongside uninfected human fibroblast DNA which had been

spiked with 3.3to825pg ofpBam 1,correspondingto1 to250

HSV-1 genomecopies per cell in 2.5 ,ug of human fibroblast

DNA. The digested DNAs were separated by agarose gel

electrophoresis and analyzed by Southern blotting with

BamHI-lasaprobe. An example of this is shown in Fig. 5c. In

some cases, the blot was reprobed with a human DNA

sequence (human 1-galactosidase cDNA) to normalize the

totalamountof DNA loadedand transferred in each lane,but

itwasusually found tobe quite constant. Titers in PFU had

been determined in the presence of HMBA, which has been

shown previously by McFarlane et al. to help overcome the

replication defect in in1814, so that in1814 can be titrated

almost as efficientlyaswild-type virus (39). The ratios of IGU

to PFU in one experiment were as follows: D30HA3, 5.4;

14HA3,20.3; and 14HRA3, 3.2. Although the difference in the IGU/PFU ratio between D30HA3 and 14HA3 was fourfold in thisexperiment,thedifference was only twofold in an indepen-dent experiment. Some degree of variability in virus titer is inevitable, in part because of differences in the growth state

and passage number of permissive cell lines. Since MOI is a

verycritical parameterfor studying cytotoxicity, we have tried

tominimize the effects of variation in virus titer by comparing

the effects of different viruses using titer values that had been

determined at the same time. In some of the experiments

described in this paper, we have compared the toxicity of

different virusesusing PFU rather than IGU as a measure of

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CYTOTOXICITY OF REPLICATION-DEFECTIVE HSV-1 MUTANTS 6351 virus titer, but we bear in mind that this places more stringent

conditions on the VP16 mutants because of at least a two- to fourfold impairment in plaquing efficiency.

Cell viability measurements were also determined by trypan blue exclusion, as follows. Human primary fibroblasts were

seeded onto 24-well plates at a density of 5x 104cells per well

and infected in triplicate the next day with mutant virus strains

at an

MOI

of 10 PFU per cell. Viruses that were inactivated by

UV irradiation were exposed to a constant UV source (1,200

[LW/cm2)

for 5

min.

We had previously determined that this

UV dose would reduce

,-galactosidase

expression in CgalA3

by more than 30-fold and reduce virus titer by more than 100,000-fold (29). One hour after infection, the virus inoculum was removed and replaced with fresh medium containing full

serum, and the cells were incubated for 3 days at

37°C.

The

cells were then harvested by trypinization and stained with 0.5% trypan blue in PBS. The number of viable cells which excluded trypan blue were counted with a hemacytometer.

Superinfection rescue procedure. The ability of different mutant virus strains to persist in infected primary fibroblasts and be "rescued" by a complementing mutant strain 11 days later was measured by a semiquantitative superinfection rescue procedure similar to that described previously (29). Briefly, human fibroblasts in 10% fetal calf serum were seeded onto a

24-well plate at a density of 6x

104

cells per 16-mm well. After

having reached confluency (about 2.5x

105

cells per well) in 3

days, cultures on duplicate plates were infected in duplicate

with each mutant virus strain, at an

MOI

ranging from 0.005 to

2.5 PFU per cell. After 1 h, the cells were washed three times

and either maintained in 0.2% serum for 11 days (with fresh

medium added every 2 to 4 days) or processed directly for the superinfection rescue. The rescue was performed by

superin-fecting cells with the

ICP8

mutant d301 at an

MOI

of 5 PFU

per cell in the presence of 5 mM HMBA for 4 h. The superinfected cells were then harvested by trypsinization, and appropriate dilutions were plated onto 10-cm plates preseeded

with

E5

cells (to titerIE 3 deletion mutants) or V9 cells (to

titer UL42 deletion mutants), in 5 ml of medium containing 5 mM HMBA and 10% pooled human serum. After 16 h, 5 ml of additional medium was added, and the cells were incubated at

37°C

for 3 to 4 days until the resulting plaques could be

counted. In control experiments, the highest number of plaques for a replication-defective mutant obtained on day 0 without superinfection was 3.4% of the number of plaques obtained with superinfection, indicating that the vast majority

of plaques which do arise do so through complementationwith

the superinfecting virus and not because of persistence of viable intact viral particles (26). By day 11, no plaques were

observed without superinfection. Also, no more than one or

two plaques have been detected following superinfection of mock-infected cells; these few plaques probably represent

occasional rescue of the

ICP8

deletion in d301 during

prepa-ration of virus stocks. Previous analysis has shown that the

plaques which arise from the superinfection rescue procedure

represent a mixture of the initial virus persisting in the cells

and the superinfecting virus (29).

RESULTS

Inhibition of colony formation with transfected HSV-1

se-quences. To determine whether we could identify potential

virus genes responsible for cytopathogenicity, we cotransfected

BHKTK- cells and CV-1 cells with plasmids containing each

of the candidate viral genes expressed by an IE 3 deletion

mutant (Fig. 1) together with pSV2neo in a 10:1 ratio (wt/wt).

The cells were then split at various dilutions and placed under

selection for neomycin resistance, and the number of colonies obtained was counted 10 to 14 days later. For the sake of completeness, we also examined the effect of IE gene 3 in these experiments. The plasmids containing the IE genes were chosen or constructed so that the presence of additional open

reading frames or the presence of extraneous sequenceswould

be very limited. However, the frequency of colony formation could be influenced by promoter competition, the presence of

"poison"

sequences, or additional open reading frames in the

cotransfected test plasmids. To control for these

possibilities,

we also examined the frequency of colony formation with

controlplasmids containing mutations in the coding regions of

each of the respective IE genes. To ensure that the plasmids

encoding IE genes 1, 2, and 3 expressed the expected IE

proteins in transfected cells, we also performed Western

(immunoblot) analysis with specific monoclonal antibodies

raised against these IE proteins (Fig.1C).In additionalcontrol

experiments with luciferase as a reporter gene, we have

determined that the promoters of IE genes 1, 2, 3, and 4/5were

highly active in transfected BHK TK- and CV-1 cells (26).

As shown in Fig. 2, plasmids encoding IE 1 (plll), IE 2

(pIE63

and pIE63+), and IE 4 (pIE68) all strongly inhibited

the frequency of colony formation in both BHK TK- andCV-1

cells, in comparison to the number of coloniesobtained by the

control cotransfection with pBluescript KS. Cotransfection

with control plasmids bearing lesions in thecoding portions of

IE genes 1 (pllOdel4), IE 2 (pXin63+ and pX63.1),and IE 4

(pZ4) reduced the inhibitory effects observed with the intact IE

genes. The inhibitory effect of IE 2 was more completely

abolished by pX63.1, which has a 28-bp deletion in the IE 2

coding region, than by pXin63+, which has a singleXhol linker

insertion mutation. Unexpectedly, both plasmids encoding IE

3 (p175 and pKl-2) strongly inhibited colony formation in

CV-1 cells but not in BHK TK- cells. Theinhibition of colony

formation in CV-1 cells cotransfected with IE 3sequences was

more effectively abolished by pD8, which encodes an IE 3

deletion product retaining transactivation activity butdefective

for repression (48), than by pD9, which encodes an IE 3

product with full repression activity but only 4% of the

transactivation activity of the wild-typeIE 3 protein (48). The

plasmid encoding IE 5 (pIE12) did not cause inhibition of

colony formation in BHK cells, although amoderate inhibition

of transformation frequency (50%) was observed inCV-1 cells.

Inhibition of colony formation following cotransfection with

plasmids encoding IE genes 1, 2, and 4 has alsobeenobserved

with a different selectable markerconstruct, in which theneo

gene was driven by the human HPRT promoter instead of the

SV40 promoter (data not shown). We have also determined

that neither plasmid pKHF, which encodes thelarge subunit of

ribonucleotide reductase (ICP6), which is alsoexpressed asan

IE gene, nor pKHFA, which harbors adeletionwithin the ICP6

coding region, causes inhibition of colony formation (26).

Finally, since the products ofIE genes 1, 2, and 3areknownto

modulate transcriptional activity, it was possible that they

might directly affect the level ofneomycinphosphotransferase

produced by the selectable markerplasmid, therebyproviding

a trivial explanation for their effect onthefrequencyofcolony

formation. We therefore measured the effect of

cotransfecting

IE genes 1, 2, and 3 with the neo gene on theresultinglevel of

neomycin phosphotransferase activity and determined that

while plasmids bearing IE 2 or IE 3 had little effect,IE 1

caused a moderate (twofold) stimulation in activity (data not

shown).

Isolation of mutant viruses. (i)IE 3-VP16 double mutant.

To reduce the expression of HSV-1IE genesduringa

nonpro-ductive infection, we have constructed double mutant viruses

VOL. 68, 1994

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(U

0

Ist

IE gene: 1 1-1 2

1

21

2-1

3 | 3

1

3-1 3-1 4

1

4-1 5

1

5-|

o-

_X N 0.

v-0 0

4(

0. " CL

06

CL~~~~0

ffi $ CL 3R^Ssa f S ^

Plasmid

FIG. 2. Effect of HSV-1 IE gene-carrying plasmids on the

fre-quencyofcolonyformation in cotransfected BHKTK-andCV-1 cells.

Approximately 105cellsper35-mm wellwerecotransfected in

tripli-cate with 0.25 ,ugofpSV2neoand 2.5 ,ugof the indicated testplasmid.

Cellswerewashed after24 handsplitthefollowing dayat 1:4 and 1:50 dilutions into selectionmediumcontaining G418(400 ,ug/ml). Colo-nieswerecounted 10 to 14daysafter transfection. Anaverage of 933

and 2,916 colonies per 0.25 ,ug of pSV2neo were obtained in the control transfections(with2.5 ,ugofpBluescript KS)of BHKTK-and CV-1 cells, respectively. No colonies developed in the absence of pSV2neo.Errorbars indicate standard deviations.

withamutation inanessentialgene toprevent lytic replication

and with theinsertionmutation in the VP16genedescribedby

Preston and colleagues (2) to abolish the ability of VP16 to transinduce IE gene expression. The double mutant 14HA3

wasgenerated by introducingadeletioninto bothcopiesofthe

IE 3gene of the VP16 mutant in1814. As shown in Fig. 3c,

plasmid pIllH,usedtogeneratethe IE3deletion,containsa

DNAfragment from pUC19 (a 587-bpHaeIIIfragment

con-tainingthe 5' portionof the ampicillinresistancegene) atthe

site ofthedeletiontoserve as atagto aid theidentification of viral recombinants by plaque hybridization. This plasmid is otherwise identicaltopdelIll,whichwasused togeneratethe IE 3 deletion mutant D30EBA (49). Since initially we were

unsure how well an IE3-VP16 mutantwould grow, we also isolated a derivative ofD30EBA from plasmid pIllH,

desig-natedD30HA3,toprovide apositivecontrol for identification

ofrecombinantsby plaque hybridizationwith thepUC19DNA

fragment and to ensurethatrecombinants could beidentified

under conditionsthatdid not requireselectionagainstthe IE

3 gene. Southern blot analysis of the genomic structure of

14HA3 isolates is shown in Fig. 3d (leftmost panel). The

presence of the 0.52-kb band in BamHI-EcoRI-digested

14HA3 DNA, which has been probedwith a DNAfragment

from the 5' end of IE3,indicates that theanticipateddeletion

ofthe IE 3 gene has takenplace, althoughoverexposureof this

blotdid reveal the minor presence ofa1.8-kbband,indicative

of thewild-type IE3BamHIfragment. The viral stock from

which this DNAwas prepared was also able to form a few

plaquesonVerocells,whicharenoncomplementaryfor IE 3 mutants. Therefore, this 14HA3 virus isolate was further plaque purified until no such IE 3-independent plaques ap-pearedinan inoculumcontainingat least 105PFU.

Toensurethatphenotypicdifferences between 14HA3 and

anIE3 deletionmutant wereduetothe mutation in the VP16

gene and not to a second mutation, the VP16 lesion was

repaired byrecombining14HA3 DNA with

plasmid

pMC1 (7),

which contains an intact VP16 gene. Rescued virus could be

obtained readily, since it was able to grow much more

effi-ciently than the 14HA3 double mutant in the absence of

HMBA.The genotypes of three such isolates wereexamined

by Southern blot

analysis

for the presence ofan intact 8-kb

BamHI-f fragmentin

place

of the 3- and 5-kb bands associated

withthe BamHI linkerinsertion present in in1814 and 14HA3

(Fig. 3d,secondpanel),andastockpreparedfromoneof these purifiedisolateswas

designated

14HRA3.

(ii) UL42-VP16 doublemutant.Toexamine the effect of the

VP16 mutation on virus-induced

cytotoxicity

in the

back-groundofa mutantblocked for

replication

at astepin thelytic cycle different from that ofthe IE 3 deletion mutant,wealso

introduced a deletion into an essential early geneofa VP16

mutant(seeMaterialsand

Methods).

Theearlygenewechose to mutate was UL42, which encodes a 65-kDa accessory

function for the HSV-1 DNA polymerase

(18),

essential for

virus replication

(27,

37). The purified UL42-VP16 double

mutantisolatewas designated inl850A42.

(iii) IE 3-vhs double mutant and IE 3-vhs-VP16 triple mutant.To assessthe role ofthe vhs function in the cytotox-icity of replication-defective mutants, we introduced an

HCMV-lacZcassetteinto the UL41coding regionof D30EBA

and14HA3(seeMaterials andMethods).Thegenomic struc-tures of the recombinants, designated A3vhsZ and 14HA3 vhsZ, respectively, were examined by Southern blot analysis (Fig. 3d). DigestionofA3vhsZand14HA3vhsZ DNA with SstI

gave rise to a 3.2-kb band detectable with a probe from a

BamHI-SstI subfragment ofBamHI-i, which is the size

ex-pectedfromcorrectinsertionofthelacZgene,instead ofthe

2.2-kbfragmentfromanintact UL41 5'region (Fig.3b, andd).

When this blot was reprobed with lacZ, 3.2- and 3.5-kb

fragments were detected, indicating correct insertion of the

lacZ gene into theUL41 codingregion. We also checked for

the presence of the BamHI linker in the VP16 gene of 14HA3vhsZ (but not

A3vhsZ),

indicated by cleavage of the 8-kbBamHI-f fragmentinto 3-kband 5-kb subfragments (Fig.

3d).

Smibert and Smiley have shown that insertion of an

ICP6-lacZcassetteinto theBamHIsite of the UL41 gene of an

HSV-1 KOS strain resulted in inactivation of the vhs function

(61). Since their mutation disrupted the UL41 open reading

frame afterresidue342, and in ours it is disrupted after residue

237

(see

Fig. 3b),it isprobable that insertion of the lacZ gene

at the NruIsite will also result ininactivationofthe vhs

func-tion.

Growth characteristics of mutant viruses. The insertion

mutation inin1814preventsVP16 fromforming the

multipro-tein complex that normally transactivates transcription of

HSV-1 IE genes uponinfection, thereby impairing the ability

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[image:6.612.59.296.74.407.2]

CYTOTOXICITY OF REPLICATION-DEFECTIVE HSV-1 MUTANTS 6353 a)

LAT 10 kb

UL UL42

vhs VP1 6

(UL41) (UL48)

Bamf s Ift

BamHti BamHIf

b) 1<-2.2kb --I

S B Nr Sh S S

11 I I I

C)

S Ba

LAT IE3

-*+ us -+

IE3

H

BamHlz 7.8kb

1- -1j

BB BE

II I I i,

4Evhs(iJLgq MM2

i j pBam-i-Sst

--- S E*522bp.-B

| t

--iig

lacZ L pUL41-lacZ t i a, i pIllH

c 3.5kb 3.2kb

-Sstl fragments probe

8--s4.0

5-4-4_ __

1.8-..

> N > N

" 0 ±7 C)~ I ¶2 0r

.. 3s 2

2.2-..

probe-BamHI

i-Sstifrag.

probe.

acZ

[image:7.612.154.477.75.423.2]

probe. I1E3 5' end

FIG. 3. Construction and analysis of genetic modificationstothe HSV-1genome.(a)Mapof the HSV-1genome,consisting of the unique long

(UL)andunique short (Us)regions, bound by terminal and internal long (TRL and IRL) and short (TRS and IRS) repeats. Shownarethe

locationsofgenesUL41 (vhs), UL42, UL48 (VP16), IE 1,IE3, and theLATtranscripts, and the locationsof BamHI fragments i,f, andzused

intheconstructionand/oranalysis ofmutants.(b)Construction of vhsmutants.AnHCMV-lacZcassettewasinsertedinto the NruI site of UL41, containedonplasmid pBam-i-Sst, creating pUL41-lacZ. The sizes of novel and wild-type SstI fragments detected by BamHI-iorlacZprobesare

given. (c) Deletion of the IE 3gene.Theposition of the IRScopyof theIE3transcript (line) and coding region (hatched box) isshown,asis the

extentoftheIE3deletionwithinplasmid pIllH. pIllHwasderived from pdelIll (49) by insertion ofa522-bp HaeII fragment from pUC19,to

serveas atagforthe detection of recombinantsbyinsitu plaquehybridization. OnanEcoRI-BamHI digest of unpurified recombinant viral DNAs,

theindicatedIE35' probewilldetecta 1.8-kbwild-type fragmentor a522-bp novel virus fragment. (d) Southern blot analysis ofrecombinant viruses. Firstpanel: 14HA3, D30HA3, D30EBA, and in1814 viral genomic DNAsweredigested with EcoRI and BamHI and probed with theIE 35'-end fragmentshown inpanelc.Thepresenceof the0.52-kbbandin14HA3,D30HA3, and D30EBAindicatesthatdeletion of theIE3gene

hasoccurred.Secondpanel:ViralDNAs fromin1814,D30HA3, 14HA3,and threeisolates of 14HA3 which had beenrescuedwithanintact VP16 gene(14HRA3)weredigestedwith BamHI andprobedwith theBamHI-ffragment.Thepresence of the BamHIlinkerinthe VP16generesults incleavageof the8-kbBamHI-f fragmentinto5-kband3-kb subfragments. Thirdpanel: 14hA3vhsZ, A3vhsZ,and 14HA3 DNAsweredigested with SstI.Thepresenceof theHCMV-lacZcassetteisindicatedbyanovel3.2-kbbandinplaceofthewild-type2.2-kbband whenprobedwith theBamHI-i-SstIfragmentshown inpanelb.Reprobingwith lacZgaverisetoanadditional 3.5-kb bandinthe vhsmutants.Fourthpanel:The indicatedviral DNAsweredigestedwithBamHI andprobedwithBamHI-ftoconfirm thepresenceofthe VP16 mutation in14HA3vhsZ,indicated bythe5- and 3-kbsubfragmentsofBamHI-f.Restriction sites: S,SstI; B, BamHI; Sh, SphI; Ba, BalI; E, EcoRI; Nr, NnrI.

of in1814toinitiate infectionatlowMOI(2).In thepresence

ofHMBA,however, in1814 is abletoinitiateinfection almost

as efficientlyaswild-type HSV-1 (39). To determine whether

thenewVP16mutantvirusesexhibit asimilar phenotype, we

measured theyieldofprogenyvirusafter24h ofinfectionof

permissive cells at a low MOI (0.01 PFU per cell), in the

presenceandabsence of HMBA. Asshown in Table 1,viruses with the VP16mutation(14HA3,14HA3vhsZ,andin1850A42)

grew very poorly in the absence of HMBA, but their yields

were improved by30- to 60-fold in itspresence. HMBA had

little effect on the yield of viruses without the VP16 lesion

(D30HA3, 14HRA3, A3vhsZ, andCgalA42). Viruseswith the

UL41 mutation were further impaired for growth, since the

yield of A3vhsZ was 10-fold below that of D30HA3, and

although in this experiment the yield of 14HA3vhsZ with

HMBA was only 2-fold below that of 14HA3, the titers of stocks of14HA3vhsZwere usually5- to 10-fold below thatof 14HA3 (26). These observations are consistent with those of other studiesreporting that large insertion or deletion muta-tions in the vhsgenecause a5-to10-folddropin viral titer(15,

61). Togetherwith the confirmation ofgenomic structure by

Southern analysis,we conclude from these observations that d) ) ,n< <

tx0

- I 14HR.A3

C :1 1 2 3

.l

-C C to _1.V.m>

J=

9 A 4 x

0 < - 11-1

0.52-.*11..M

probe:BamHlf

*e **O-m

.4-8 -5 -4-3

probe. BamHIf

VOL.68, 1994

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TABLE 1. Yield ofprogeny

Yieldb Ratio, YieldtoD30HA3orCgalA42d

-HMBA +HMBA +HMBA/-HMBAC -HMBA +HMBA

D30HA3 IE 3- 1.0x 106 0.9x 106 0.9 1.0 1.0

D30EBA IE 3- 1.1 x 106 1.2x 106 1.1 1.1 1.3

14HA3 IE 3-,VP16in 3.8 x 103 2.3x 105 60.5 0.004 0.25

14HRA3 IE3- 1.5x 106 2.0 x 106 1.2 1.5 2.0

A3vhsZ IE3-,vhs-lacZ 1.0X 105 1.2X 105 1.2 0.1 0.13

14HA3vhsZ IE3-,VP16'n,vhs-lacZ 2.8 x 103 1.0 X 105 35.7 0.003 0.11

CgalA42 UL42-, BamHI-z-lacZ 1.2 x106 1.5 x 106 1.3 1.0 1.0

inl850A42 UL42-,tk-lacZ,VP16in 2.5 x 103 1.0 X 105 40.0 0.002 0.067

aGenotype indicateswhetherviruseshavedeletions inIE3 orUL42,thepresenceof the insertion mutation in theVP16genedescribedbyAceetal.(2),and the presenceof a lacZ geneinsertedinto theBamHI-zregionor alacZinsertional mutationinthe vhsortkgene.

bPermissivecells thatcomplementgrowth ofmutants(E5 forIE3mutants,V9 forUL42mutants)wereinfectedat anMOIof 0.01 PFU per cell for 20 h ineither theabsenceorcontinuouspresence of 5 mMHMBA,asindicated.Thecellswerethenscrapedinto the medium anddisruptedbyfreeze-thawing,and the titers ofvirus intheresultingsupernatants were determined on theappropriatepermissivecells in the presenceof 5mM HMBA. Valuesrepresenttheyieldof progenydetermined fromduplicate infected cultures and arerepresentativeofrepeatedexperiments.

cValuesrepresent theyieldofvirusdeterminedinthepresenceofHMBAdividedbytheyieldof virusdeterminedinthe absence ofHMBA.

dValues represent theyield of eachIE 3 mutantdividedbytheyieldof

D30HA3

ortheyieldof

CgalA42

and theUL42-VP16mutantin

1850A42

dividedbytheyield ofCgalA42, determinedin the presence and absenceofHMBA.

wehavesuccessfullygenerated virus strains with defects in the

virion components vhs and/or VP16 whicharealsocompletely

defective forreplication.

Comparison ofcellviabilityfollowinginfection withmutant

virus strains. Wehave examined the effect of infection with

the different mutant virus strainsonthemorphology of human

primary fibroblast cultures. Confluent monolayers of

hu-man fibroblasts were either mock infected or infected with

D30HA3, 14HA3, A3vhsZ, 14HA3vhsZ, CgalA42, orinl850A

42 at an MOI of 2.5 PFU per cell, and the cultures were

examineddailyfor the appearanceof CPE(Fig.4).The results

show that cultures infected with 14HA3 (Fig. 4c) remained

almostashealthyasthemock-infectedcontrol(Fig. 4a),while

those infected with D30HA3 (Fig. 4b) exhibited increasingly

severe CPE withincreased timepostinfection. Therefore,as a

result of the VP16lesion in14HA3,thecytopathogenicity of an

IE 3 deletion mutant has been significantly reduced. In

con-trast, A3vhsZ (Fig. 4d),which has a defective vhs function in

addition to the IE 3 deletion, did not appear to be any less

cytopathogenic thanD30HA3.Cultures infected with the triple

mutant 14HA3vhsZ (Fig. 4e), which has a VP16 lesion as well

asthe IE 3 and UL41mutations, also remained healthy for the

duration of the experiment. However, inl850A42 (Fig. 4g),

which has aVP16lesion and isdeleted for the essential early

geneUL42, didcausegeneralized CPE in infected cultures at thisMOI,asdidCgalA42(Fig. 4f), which is deleted forUL42

buthasanintact VP16 gene.Inthese experiments, the cultures

wereinfected with the same number of PFU, even though the

plaque-forming ability of the VP16 mutant strains (determined

in the presence ofHMBA) was likely to represent a two- to

fourfold underestimate of the relative number of infectious viral genomes (see Materials and Methods) and thereby place

amorestringent test on the ability of the VP16 mutants not to

causeCPE.Nevertheless, these results show that as a result of the VP16 mutation, the cytopathogenicity of an IE 3 deletion

mutanthas been sufficiently reduced so that an infected culture

does not develop extensive CPE when infected at anMOI of

2.5, whereascultures infected with mutantscarrying mutations

in IE 3 aloneorUL42alone,an IE 3-vhs double mutant,ora

UL42-VP16 doublemutantall developCPEatthisMOI. We

note,however, thatcells infected with14HA3and14HA3vhsZ

did exhibitsomegranularity,although this experimentwas not

designed to quantify the effects of viral infection on cell

division and/or survival.

We therefore set out to quantify the effect of the VP16

lesion on the toxicity of the IE 3 and UL42

replication-defectivemutantsas afunction ofMOI. In thisexperiment,we

used IGU as the measure of virus titer to compensate for

differences in theefficiencyofplaquingbetweenVP16mutants

andVP16-intact virus. The determination of IGU titer for the

stocks of IE 3mutantsused inthisexperimentis shown inFig.

Sc and Table 2.To assaytoxicity,wecountedcell numberat3

days postinfection, to allow sufficient time forvirus-induced

cytotoxicity to occur. We used a mitotic fibroblast cell line

because CPE occurs faster and there is less ambiguity in

distinguishing livingcells than ifwehad usednondividingcells.

Cell numbertherefore reflects both the survival andreplication

of cellsoverthe3-dayperiod after the infection. Subconfluent

monolayersof humanfibroblastswereinfectedwithD30HA3,

14HA3, 14RHA3, CgalA42, and inl850A42 over a range of

MOIs from0.1to25 PFU percell. Thetiters of the virus stocks

used inthisexperimentweredeterminedatthe same time in

termsof IGU per mltocompensatefor the reducedplaquing

efficiency of the VP16 mutants(asshownfor the IE 3 mutants

in Fig. 5c and Table 2). As shown in Fig. 5a, the curve

produced from the number ofadherent cells at 3 days

postin-fection with increasing amounts of 14HA3 is shifted

signifi-cantlytothe right of the curves corresponding toD30HA3and 14HRA3, indicatingthat14HA3is less toxic over a wide range

ofMOIs.Inthecaseof14HA3, the curve was steepest after an

MOI of 10 IGU per cell,which indicates the point at which

virustoxicity is measurably affecting cell growth and survival,

whereas for infections with 14HRA3 and D30HA3, significant

loss in cell number occurred after an MOI of approximately 3

FIG. 4. Infection ofhumanprimaryfibroblastswithHSV-1 mutants. Confluent monolayers of human fibroblasts were either mock infected (a) orinfected withD30HA3(b),

14HA3

(c),A3vhsZ(d), 14HA3vhsZ(e),CgalA42(f), orin1850A42(g) at anMOIof2.5PFU per cell. Monolayers wereobserveddailyfortheappearanceofCPE andphotographed.

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[image:8.612.61.553.86.203.2]

~~~~~~~

_

2 day

3das

4 ay

6355

1

day

on November 9, 2019 by guest

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-u!

44-1

0

lL

(-0.

LC.

la

Cu

en

'a-co

w

S.

a6

Multiplicity of infection ("infectious genome units" per cell)

c)

re

LIC]c

et US)0L 0LO

FIG. 5. Effect of the VP16 mutationon cell survival and growth

after infection with mutant viruses. Subconfluentmonolayersof

hu-manfibroblasts (5 x 104per16-mmwell)were infectedin triplicate

with(a)D30HA3,14HA3,or14HRA3or(b) CgalA42orin1850A42 at increasingMOIs.The MOI is shownhere in terms ofIGU per cell.

TheIGU titersof virus stocksweredeterminedasdescribed forpanel c.Theaverage number of adherentcellsremainingat 3days postin-fection,determined with aCoultercounter,are plotted againstMOI (errorbarsindicate standarddeviations).T50is definedasthe MOI at

which the number of adherent cells was reduced by 50% of the

maximumnumberof cellson acontrol uninfectedplate(IGUpercell). (c)Southernblotshowingtheamount of viral DNAdetected inhuman

fibroblasts infected with 25-pAl aliquots of D30HA3, 14HA3, and

14HRA3. Theinfected-cell DNAs and cellular DNAspikedwith the equivalent of 0 to 250 copiesofpBaml plasmidDNAper cellwere digestedwith BamHI and probed with radiolabeled BamHI-l

frag-ment. Thecalculationof IGUfromdensitometricanalysisof thisblot

is shown inTable 2.

IGU per cell. Takingthe maximum number ofcells from a

controluninfected wellatday3 tobe300,000,the titer(inIGU

percell)atwhichcellnumberwasdecreasedby50%(T50)was

30 for 14HA3, 9for D30HA3, and4for 14HRA3. Therefore,

using theT50 valuesdeterminedfrom this assayas ameasure

[image:10.612.63.306.76.474.2]

of virus toxicity, the IE 3-VP16 double mutant 14HA3 is

TABLE 2. Infectious genome titer

Virus _copies/celraNo. of IGUb PFUc IGU/PFU ratio

D30HA3 45.3 1.8 x 109 3.5 x108 5.1

14HA3 32.3 1.3 x109 6.4 x 107 20.3

14HRA3 20.0 8.0 x 108 2.5 x 108 3.2

aThe number of viral genome copies percell was determined following infectionof106 TO-119 fibroblastswith 25 ,ul ofeach virus stock and from densitometry of the Southern blot showninFig. 5c,asdescribed in Materials and Methods.

b The titer of virus in infectious genome units (IGU) per milliliter was calculatedfrom thecopy numberof viralgenomespercell(N)multipliedbythe total numberof cells(106)and dividedbythevolume ofthevirus inoculum(V, inmilliliters)asfollows:(Nx 106)/K

cThetiter ofvirus in PFU per ml wasdetermined from thesamevirus stocks atthe same timefollowinginfectionofE5cells in thepresenceofHMBA.

betweenthree- and sevenfold less toxic than either of the IE 3

mutantswhich haveanintact VP16 gene. Since intwoseparate

determinations 14HA3 had a two- to fourfold-higher IGU/

PFUratio thanD30HA3,itfollows that if PFUwasusedasthe

measure of virus titer in this cell-killing experiment, the

reductionintoxicityasaresult of the VP16mutationinthe IE

3 mutantbackgroundwould be lesspronouncedor not

appar-ent.

Incontrast,cell survivalfollowing infection with inl850A42

was not markedly improved over that after infection with CgalA42 (Fig. 5b). A steep reduction in cell number was

apparentat anMOI of 2 IGU per cell for both UL42mutants.

The titers atwhich the maximum cell number (300,000) was

reducedby50%were2IGU percell forCgalA42and 3 IGU

per cell for inl850A42. Therefore, the VP16 lesion in

in1850A42appearedtohave little benefitover aUL42 deletion

alone in reducing viral toxicityin this assay. In addition, the

UL42mutants were moretoxic than the IE 3deletion mutants, irrespectiveof the VP16 lesion. However, these results donot

rule out the possibility that inl850A42 is less toxic than

CgalA42 at much lower MOIs, as examined in the following experiment.

Rescue of virus from persistently infected cells. We have

previouslyusedanin vitrolatency system to show that at a low

MOI,asmall percentage of cells infected withanIE3deletion

mutantmaintain the virus inabiologically retrievable form for

at least2 weeks (29). However, it was notpossible to

signifi-cantlyincrease the number of cells which could maintain virus by increasingthe MOI,presumably because of increased viral toxicityathigherMOIs. We reasoned that if any of the mutant virus strains described in this study have reduced toxicity, cells

should be abletosustaininfection at a higher MOI and more

virus should be available for rescue a week or later after

infection. To test this hypothesis, we infected human

fibro-blasts with D30HA3, 14HA3, CgalA42, inl850A42, A3vhsZ,

and 14H3vhsA3 at increasing MOIs. We used PFU as the

measure of virus titer inthis experiment since the method of

titer determination will not ultimately affect the maximum

number of virus that can be rescued over a wide range of

MOIs. The amountofvirus that could be recovered

immedi-ately(1 hpostadsorption) or 11 days after infection by rescue

with a complementing HSV-1 strain deleted for ICP8, d301

(19),is shown inFig.6. Superinfection with d301 provides the

IE 1 geneproduct, VmwllO,which is important for

reactiva-tion oflatent virus(34, 57),aswellasthe IE 3 and UL42 gene

products, which are needed for complementation of viruses

with mutations in these genes. Since the plaques from the

rescueprocedure have arisen from infected human fibroblasts

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[image:10.612.319.563.83.141.2]

CYTOTOXICITY OF REPLICATION-DEFECTIVE HSV-1 MUTANTS 6357 6

5 4 3

I

0._L

0

a

I-z 2 1 5 4

3 2

5

5 4 3 2

2 3 4 5 6/2 3 4 5 6

Number of

infecting virus

(log

PFU per

well)

FIG. 6. Recovery of virus mutants from infectedfibroblasts. Confluent monolayers of human fibroblasts (approximately 2.5 x 105cellsper 16-mmwell)wereinfected at MOIs of from 1 X 103to5 x 105PFU perwell with each of the mutantsD30HA3, 14HA3, CgalA42,in1850A42, A3vhsZ, and14HA3vhsZ,asindicated. Graphs show the average number of plaques from duplicate cultures that could be recovered 1 h (open symbols)or11days(solid symbols) postinfection.Recovery wasperformed by superinfection with theICP8mutantd301(19). Superinfected cells wereplatedoutatvariousdilutionsontomonolayers ofE5cells (for IE 3mutants)or V9cells(forUL42mutants) as appropriate and incubated at37°Cinthepresenceof5%humanserumuntil plaquescould be counted.

platedonpermissive cellmonolayers andnotfrom freevirus,

theyshouldnotexceed the total number of cells available for

infection, which was approximately 1 x 105 to 2 x 105, as

determined by counting with a Coulter counter. Therefore,

since cells whichwereinfectedbymorethanoneviruscanstill

give rise to only one plaque, theactual amount ofpersistent

virus in this experimentcannotbe determined above a

maxi-mumof1PFU percell. In somecases, the number of rescued

plaquesatday0 exceeded the number ofPFUin theinfecting

inoculum (for instance, 1 x 103 PFU ofD30HA3 resulted in

2.5 x 103 rescued plaques), which probablyreflects the fact

that theactual titer of infectious genomes abletocomplement

d301 isseveralfold higherthan the PFU titer.

Asshown inFig. 6, infection with eitherD30HA3or 14HA3

at increasing MOIs led to a maximum number of plaques

obtainable on day 0 of approximately 105, which correlates

reasonably well with the total number of cells available for

infection. Striking differences were observed between the

numbers ofD30HA3and14HA3which could be rescuedatday

11.First,atlowMOIs(103to104PFUper105cells),lessthan

10% oftheinput D30HA3 could berecovered,comparedwith

45 to 50% for 14HA3. Second, the maximum number of

rescuableD30HA3 reachedasharppeakof 3.5 x 103 PFUat

an MOI of 104 PFU per well and then rapidly declined at

higher MOIs. In contrast, the number of rescuable 14HA3

began toplateau at 9 x

103

PFU at an MOI of

104

PFUper VOL.68, 1994

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well and reached a

peak

of 1.2 x

104

PFU at an MOI of 105

PFU perwell. The number of rescuable 14HA3 declined above

an

infecting

inoculum of

105

PFU perwell.

Therefore,

by

the

following

two

criteria,

(i)

the

proportion

of

infecting

virus that

can be recovered at a later time

point

and

(ii)

the maximum

amountof virus thatcanbe recovered after sustained infection

of

cells,

14HA3 has a

considerably

less toxic

profile

than

D30HA3.

Comparison

of the UL42 mutants

CgalA42

and

in1850A42 revealed that a

greater

proportion

of in1850A42

than of

CgalA42

could be recovered at low MOIs.

Also,

the

maximumamountofin1850A42 that could be recovered

(1

x

104

PFU)

was

substantially higher

than the maximumamount of

CgalA42 (6

x 102

PFU).

However,

above an

infecting

inoculum of

104

PFU per

well,

the recovery of both viruses

dropped precipitously, indicating

that both UL42 mutants

weremore toxic thantheir

corresponding

IE 3 mutant

coun-terparts,

in

agreement

withthe results described earlier.

Com-parison

of the IE 3-vhs double mutants A3vhsZ and

14HA3vhsZ

showed

again

that inclusionof the VP16 mutation

(in 14HA3vhsZ)

allowed an increased recoveryof virus at 11

days postinfection,

intermsof both the

proportion

of

infecting

virus recovered

(less

than 10% for A3vhsZversus 30 to 50%

for

14HA3vhsZ)

and themaximum sustainable virusload

(3

x

102 PFU for A3vhsZ versus 1 x 104PFU for

14HA3vhsZ).

Interestingly,

A3vhsZ

appeared

tobemoretoxicthan

D30HA3

by

this assay. The recovery ofA3vhsZwas less efficient than

that of

D30HA3

on

day

0,

and the

peak

amountofA3vhsZthat

could be recoveredwas 10-fold below that for D30HA3. In

terms of virus recovery and maximum sustainable virus

load,

the

triple

mutant

14HA3vhsZ

wassimilartothe doublemutant

14HA3,

indicating

that inclusion of the vhs mutation in this mutant

background

had littleor no benefit in

reducing

virus

toxicity.

Finally,

wetestedthe

possibility

that thevhs function does

contribute to CPE but its effects are

usually

masked

by

a

greater

level of

cytopathogenicity

caused

by

viral gene

expres-sion. We therefore infected human

primary

fibroblasts at an

MOI of 10 PFU per cell with

D30EBA,

A3vhsZ,

and

14HA3vhsZ

whichhad either been untreatedorirradiated with UV

light

toabolish viral gene

expression.

Ithas been shown

previously

thatthe vhs function isnotinactivated

by

suchUV treatment

(16, 44).

Cellswereharvested

by

trypsinization

at3

days

postinfection,

and cell

viability

wasdetermined

by

count-ing

cells which excluded trypan blue

dye (Fig. 7).

In the

absence of UV treatment, the number of viable cells in

comparison

tothat inamock-infectedculturewasreducedto

less than 3% for A3vhsZ and D30EBA and to 15% for

14HA3vhsZ.

After UV irradiation ofthese mutants, cell

via-bility following

infectionwas much

improved

but very similar for each mutant

(about

42%), indicating

that factors other thanvhsare

causing

cytotoxic

effects in the absence of gene

expression.

In a

previous

study,

we found that cells infected

withstrain KOS and the vhsmutantof strainKOS called vhsl

(53)

suffered

cytopathic

effects at an MOI of2, even in the

presence of

phosphonoacetic

acidtopreventviral DNA

repli-cation,

whereas infection with UV-irradiated viruses at the

same

MOI

abolished CPE

(29).

We therefore infected cells

with UV-irradiated KOS and vhsl at an MOI of 10 to determinewhetherdifferencesin theinduction of CPE can be detected at a

higher

MOI.

Cell

viability

following infection

withthese viruses intwoseparate

experiments

wasreduced to

between 52 and 62%

(Fig. 7),

and therewasnot a significant

difference between vhsl and

KOS, indicating

that the vhs function of strain

KOS,

like that of strain 17+, does not contributein a

major

waytothe induction of CPE.

0

-4a₀

0 v

.0 DrI.

m -I .< N N N N

0 ciO r i E a A s s

Exp.I Exp. 2 i = _

Virus strain

FIG. 7. Effectof vhs mutationoncellviabilityafter infection with

UV-irradiated viruses. Humanprimary fibroblastswere seeded at a

densityof 5 x 104cellsperwell and infected intriplicatethefollowing

day with KOS, vhsl, D30EBA, A3vhsZ,or 14HA3vhsZat anMOI of

10 PFUpercell. At 3 days postinfection,the cellswereharvestedby

trypsinization, and the number of viable cells in each culture was

estimated after staining with 0.5% trypan blue and counting the

unstained cells. The average numberof viable cells is shownas the

percentageofviablecells inamock-infectedculture,determinedfrom

counting in eight fields ofahemacytometerfor each of threeinfected

cultures. Error bars indicate standarddeviations.

DISCUSSION

Replication-defective mutants of HSV-1 retain a strong

potential to induce CPE andcausecelldeathduringinfection

of nonpermissive cells. The results of a previous study had

indicated that viral gene expressionwas amajor factor

causing

CPE (29). In an attempt to construct rather more

benign

vectors for gene transfer, we have in this study examined

individual HSV-1 genes and virion components which mightbe

responsiblefor CPE. We have shown thatatleastthreeHSV-1

genes, which are efficiently expressed by an IE 3 deletion

mutantduringinfection ofnonpermissive cells, cause a marked

inhibition ofcolony formation in cotransfection experiments.

As a means to reduce IE gene expression globally in a

replication-defective

mutant,andasan alternative to trying to produce a mutant defective for three or four IE genes, we

constructed viruses which were deleted for either of the

essential genes IE 3orUL42 andincluded the mutation inthe

VP16 gene ofin1814 describedby Ace et al. (2). This mutation

abolishes the ability of VP16 to transinduce IE gene

expres-sion. The IE3-VP16 double mutant 14HA3 is significantly less

cytotoxicthan a single mutant deleted for IE 3, whereas the

reduction intoxicityof theUL42-VP16 mutant is less marked.

The vhs function has also been considered a potentially

cytotoxiccomponentof the HSV-1 virion. However,inclusion

ofamutation in the vhs gene does not reduce the

cytopatho-genicity of either an IE 3 deletion mutant or the double IE

3-VP16 mutant.

Virally encoded cytopathogenic gene products. Plasmids

bearing

HSV-1 IE genes 1, 2, and 4 had a marked inhibitory

effect on the

ability

to obtain colonies from a cotransfected

selectable marker gene (Fig. 2). Plasmids bearing IE 3 also

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[image:12.612.341.533.73.287.2]