Host range temperature-sensitive mutants of herpes simplex virus type 2.

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Vol. 18,No.1

PrintedinU.S.A.

Host Range

Temperature-Sensitive

Mutants

of

Herpes

Simplex

Virus

Type

2

D. WESTMORELAND AND F. RAPP*

Department ofMicrobiology, The Milton S. Hershey Medical Center, ThePennsylvaniaStateUniversity, College ofMedicine,Hershey,

Pennsylvania

17033

Received forpublication22September1975

Twosmall-plaquemutantsofherpes simplex virus type2(HSV-2) (strain333),

whosegrowthat39Cwasblockedincertain celltypes

(cell-dependent

tempera-turesensitivity), werecompared with theparentalvirus inanumber of biologi-cal assays. One mutant (no. 69) was found to produce a large number of morphologically normal, but noninfectious,particles; undernonpermissive

con-ditions, these mutant particles were able to interfere with the replication of

wild-type HSV-2. The other mutant (no. 74), which is known to belong to a

different complementation group, appeared to direct little virus DNA synthesis, even atthepermissive temperature. ProgenyproductionandvirusDNA synthe-sis incells infected bymutant 74 were delayed in comparison with wild-type virus-infectedcells. Bothmutants werefoundtobe moresensitiveto UV

irradia-tionthan theparentalvirus; this wasespecially marked in thecase of mutant

74. Moreover, this mutant wasfoundto haveahightransformingefficiency at

much lower doses of irradiation than those needed to abolish the cytopathic

effect ofwildtype HSV-2.

Herpessimplex viruses (HSV) are capable of

adiversity ofinteractions with a wide range of

host cells. The consequences of infection

in-cludecell death (2, 16, 35) with or without virus

progenyproduction, virus persistence in a

la-tent, nonreplicative form (8, 32, 38), and cell

transformation with very little expression of virus-specified functions (10-13, 26, 30, 40).

There is, as yet, little information about the

natureof host-orvirus-specified control

mecha-nismsthatdeterminetheresponsetoinfection.

Indeed, despiteintensive study (16-18), many of

the crucial steps in HSV replication remain

unclear. A particularly profitable means of studying virus functions is the use of

condi-tional, lethal, usually temperature-sensitive

(ts) virus mutants. Such mutants have been

successfully employedintheconstructionof the

genetic map of HSV (5, 14, 36, 37, 39), and

recently detailed examination of biological

le-sionsresponsible for thetemperature-sensitive

phenotypehasbeguninseverallaboratories(1,

3, 15). Theproducts of infection and the ability

of tsmutantstotransform cells at the

nonper-missive temperature have been areas of most

extensive effort (26, 40).

Recently, Koment and Rapp (22-24)reported

the isolation ofa group of

temperature-sensi-tive mutants with rather different properties

from thosedescribed above. The

temperature-sensitivephenotype of these mutants was only

apparent in certain types of cell, conferring

"host range" characteristics upon the group.

Virus sensitivity to high (39 C) temperature

wasdemonstratedinhamsterand mouse

fibro-blast cultures butwas much less apparent in

epithelioidcellsorhumanfibroblasts.

Believing that these mutants might provide

a tool in the elucidation of virus-cell

interac-tion, their behavior in a number ofbiological

tests was compared with that of the parental

virus. Thework described inthis

communica-tionisanextensionof earlier studieson twoof

thetemperature-sensitive host range mutants, carried out to determine more specifically the

nature ofthe defectin virusproduction under

nonpermissive conditions and to compare other biological properties of mutant and wild-type viruses.

Ofparticularinterest was the transforming

ability ofthemutants. Ithas beenestablished

inmanylaboratories that, if the cytopathic

ef-fect of HSV is destroyed, the virus has the

capacitytotransform cells from hamsters, rats,

mice, and humans (10-13, 26, 40). It was

thought that mutants whose capacity for

pro-ductivegrowth was genetically reduced might

g.g

of strepto-mycin per ml(completemedium 199). Primary HEF cells were added to plastic petri dishes (60 mm), plastic tissueculture flasks (25 cm2), or glass pre-scriptionbottles (16ounce[ca.0.473liter])as appro-priate.The cellswereused when confluent

monolay-ershadformed.

Rabbitkidney(RK)cells werepreparedfrom the kidneys of 21- to28-day-oldrabbits by the technique previously described(11)andwereseeded into plas-ticpetridishes (60 mm) inEagle mediumplus

so-diumbicarbonate,antibiotics, and fetal calfserum atthe concentrationsused with medium 199 (com-pleteEagle medium).

Human embryonic lung (HEL) cells were ob-tained from FlowLaboratories,Rockville, Md.,and were maintained in complete Dulbecco medium (Dulbecco medium plus 0.225% sodiumbicarbonate,

10%fetal calf serum, andantibiotics,asdescribed). AlsomaintainedincompleteDulbecco mediumwere

BSC-1 African greenmonkeykidneycells and 3T3-4E TK-mousecells. The latterwerephenotypically thymidinekinaseless(TK-)3T3cells obtained from Howard Green(MassachusettsInstituteof Technol-ogy, Boston, Mass.).

Virus.A strainof HSV-2designated333,isolated

from agenitallesionbyW. Rawls(whenatBaylor CollegeofMedicine, Houston, Tex.) andpassaged only in human cells, was the parental wild-type HSV-2 used in thisstudy. Two mutants of HSV-2 strain333, whose isolation andpreliminary charac-terizationhavebeenreportedrecently (22-24),were examined. Onemutant(no. 69)wasderivedfroma single smallplaqueproducedinRKcellsbya prepa-rationofwild-typevirusthathad beensubjectedto 3.6 x 104ergsof UV irradiation percm2. Thesecond mutant (no. 74)was obtained from a single small plaqueon RKcellsproduced byanHSV-2 strain333

preparation that had been grown in medium

con-taining 20 Fgg ofbromodeoxyuridine per ml as a mutagen.

Both mutants and the wild-type virus were plaque-purified four times prior to use. Virus stocks were prepared from monolayers of HEL cells that had been infected, using an input multiplicity of 0.5 PFU/cell, and weremaintained at 33 C until exten-sivecytopathic effects became apparent (36 to 40 h postinfection). At this point, the infected cells were harvested by a single cycle of freeze-thawing and were disrupted by ultrasonication, and the lysate wascentrifuged at 200xg for 10 min.The superna-tant was transferred to small vials and stored at -70C as stock virus.

Virus assay. Virus was titrated in RK cells by a plaque assay that has previously been described in detail (31). Briefly, confluent monolayers of RK cells inplastic petri dishes (60 mm) were inoculated with 0.1 mlof virus serially diluted in Tris buffer. After anadsorption period of 1 h at room temperature, 5 ml of complete Eagle medium containing 0.5% methylcellulose was added to each plate. Cultures wereincubated for 4 days at 33 C in a moist atmos-phereof 5%CO2inair, attheend of which time the cells were fixed in 5% formalin and were stained using 0.03% methylene blue solution, and virus plaques were counted with the aid of a dissecting microscope.Plaque diameter was measured with the aid of a Nikonprofile projector(model 6C) at x10 magnification; at least 200 plaques from not less than four plates were measured for each virus-cell system.Plaque "diameter" wasalwaystaken at the widest point inahorizontal direction.

Assay for DNA synthesis. The method used for DNAanalysis was similar to thatdescribed earlier by Crouch and Rapp (6). Confluent monolayers of HELcells in plastic bottles (25cm2)weretreated for 48hpriortothe experiment withcompletemedium 199,which had a reduced concentration of fetal calf serum(1%)toreduce cell DNA synthesis. The cells were then infected at an input multiplicity of ap-proximately5PFU/cellin 0.1mlof Tris buffer. After adsorption, the monolayers were rinsed with Tris buffer,and 5 ml ofcompletemedium 199 containing only2%fetal calfserum wasaddedtoeach bottle.

The incorporation of[methyl-3H]thymidine dur-ing8-h pulses wasassayed atdifferent times after infection. [Methyl-3Hlthymidine at aconcentration

of10,&Ci/ml(45Ci/mmol)wasaddeddirectlytothe

nutrient medium and, at the end of the labeling period,mediumwasdecanted from thecells, which were dislodged fromthe bottle by freeze-thawing. Cellsthathadbecomedetachedfrom themonolayer due to viruscytopathiceffectswererescued from the culture mediumbyasinglelow-speedcentrifugation andwereaddedbacktotheappropriatebottle. Har-vestedcells weredigestedfor a minimum of4h at

37C inamixtureof0.6mlof Pronase(10mg/ml);0.3

mlofSarkosyl (10% solution), and 0.3 mlofEDTA

(0.2 M, pH 8.1) in 0.015 M sodium chloride and 0.0015Msodiumcitrate (0.1x SSC),pH7.3.

Mock-infectedcultureswere alsousedtoprovide

a '4C-labeledDNAmarker in cesiumchloride gra-dients. Such culturesweretreatedbytheprocedure describedabove, except that[methyl-'4C]thymidine

at aconcentrationof0.1

;&Ci/ml

(54

;&Ci/mmol)

re-placed[methyl-3H]thymidine, and thecultureswere

93

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94 WESTMORELAND AND RAPP

incubated for24hpriortoharvestingand digestion. After digestion, 0.3 ml of[3H]thymidine-labeled

material and0.1mlof[14C]thymidine-labeled

mate-rialwereaddedto3.8ml of cesiumchloride solution in 0.1x SSC (density = 1.7936 g/cm3) and

centri-fuged inaBeckman L3-50 ultracentrifugeat35,000 rpmfor60h usinga no.40-3fixed-anglerotor.After centrifugation, 8-drop fractionswerecollected from

the bottom of the gradientontoWhatman filter pa-perdisks. Thediskswerewashed threetimesin5%

trichloroacetic acid, once in 95%ethanol, andonce inacetone;theywereallowedtodryatroom temper-ature and were placed into 10 ml offormula 949

toluene-basedscintillation fluid (New England Nu-clear Corp.). Acid-insoluble radioactivity was

countedwithaBeckman LS-250 liquidscintillation counter,whichcould distinguish thetworadioactive labels. Countsinboth channelswere printedonto tapeand the gradient profileswereplotted with the

aid of a Hewlett-Packard 9820A calculator and

9862Agraph plotter.

UV irradiationof virus. Virus (diluted in phos-phate-buffered salinetoaconcentrationof107PFU/

ml)wasirradiated in plastic dishes(60 mm)witha

Thomas UV lamp withaGE-G8T5 bulbatadistance

such that the incident dosewas about4,200 ergs/s percm2.Irradiated viruswastitrated in RKcells,as

described above.

Virustransformation assay. Theability of wild-type and mutant viruses to mediate "biochemical transformation" of 3T3-4E TK- cells to TK+ cells

wasassayed by using selection forTK+cellsin HAT

medium (7, 25, 30). Mutant and wild-type virus stockswereinactivatedbyexposuretoUV light for differentperiods of time. Trypsinized 3T3-4E

TK-cellsinsuspension (4 x 106)weretreatedwith 2 x

106PFU ofirradiated virus (calculated from preirra-diation titer) in 4.0 ml of complete Dulbecco me-dium. The mixturewasshakenatroomtemperature for 2h, afterwhich0.3-mlaliquotsweredispensed

intoeach petri dish(12by60mm)containing4mlof complete Dulbecco medium and were incubated at 37C. Within 16 to 24 hafterplating, the Dulbecco medium wasreplaced with 5 mlof complete Dul-becco containing aminopterin (0.4 ,Ag/ml), glycine (10 ,ug/ml),hypoxanthine (15jLg/ml),andthymidine

(10 gtg/ml) (HAT medium). This medium was

re-placedeverythirdday for18days and,onday 21,the

plates were fixed using 5% formalin and were

stained in 0.3% methylene blue for 20 min. After staining, colonieswerecounted and themean

num-ber of coloniesperplatewascalculated.

Assayfor defective interfering particles. (i) Con-fluent monolayers of HEF cells in bottles (16 ounce)

wereinfectedatdifferentinput multiplicities,with aconstant amountofwild-typevirus andincreasing dilutions ofmutantorwild-type"interferingvirus."

After adsorption at room temperature, the cells

wereincubatedat39Cincompletemedium 199 for

30h,atwhich timethecellswereharvested and the

viruswasextracted and titrated,asdescribed above.

(ii) A constant amount of wild-type virus and increasing dilutions of "interfering" wild-type or mutant virus were mixed in a 0.1-ml quantity,

which was then addedto confluent monolayers of HEFcells in plates(60mm).After adsorption for1h

atroomtemperature,thecellswereincubatedat33 or 39C for 4 days under complete Eagle medium

containing 0.5% methylcellulose. After4 days the cellsheetwasfixed, stained, and examined for virus

plaques, asdescribed above.

Particle counts by electron microscopy. Virus

suspensions in medium 199wereprepared for elec-tron microscopy by the technique of Monroe and Brandt (29) andwereexaminedat75kVinaHitachi

HU12 electron microscope. Virus particles were

scoredas enveloped or nonenveloped andcoredor

emptied of any electron-dense core material. All

samples for electron microscopy were blind-coded

priortoexamination.

RESULTS

Host range, temperature sensitivity, and

plaque morphology. Theresultsofplating

ex-periments in different cell types, several of

which arein accordwithpreviously published

data (23, 24),aresummarizedin Table1.Plaque

size,although heterogeneous, fellwithina

sin-gle population for each virus in each cell type. Mutants 69 and 74, which produced smaller

TABLE 1. Plating efficiencies and mean plaque size at 33 and 39 C of wild-type HSV-2 strain 333 and of

mutants 69and 74a

Wild-type333 Mutant 69 Mutant74

Hostcell Plating effi- Mean plaque Platingeffi- Mean plaque Plating effi- Mean plaque ciency39C/ size (mm) ciency 39C/ size(mm) ciency 39C/ size (mm)

33C(%) 33C 39C 33C(%) 33C 39C 33C (%) 33C 39C

HEF 10 3.3 1.3 3.3 x 10-4 1.8 4.0 x 10-5 2.0 1.7

RK 100 2.5 2.0 10 2.0 0.5 15 1.5 0.5

HEL 100 2.5 2.8 11 2.4 0.6 30 2.4 0.8

BSC-1 (African 57 0.9 0.9 23 0.6 0.5 33 0.9 0.4

green monkey kidney line)

3T3-4E TK- <10-3 1.4 <10-2 0.9 <10-3 0.9

(mousecell line)

aTheplating efficiency ofvirus stocks(grownin HEL cells) wascomparedin different cell types, using the plaque assay

technique.

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RANGE TEMPERATURE-SENSITIVE

plaques than parental 333 under all

experimen-talconditions, were found to express a

tempera-ture-sensitive phenotype that was hostcell

de-pendent. Moreover, failure of the mutants to grow at 39 C was not merely due to a prolonged

virus growth cycle since infected HEF cells

could be maintained for up to 9 days at 39 C

without adetectable increase in plaque

num-ber. Mutant virus growth at 39 C was due to

leakage, rather than reversion to wild type.

Very little wild-type or mutant virus growth

wasobserved in 3T3-4E TK- cells at 39 C.

Figure 1 shows growth curves over the first

24hafter infection of wild-type 333, mutant 69,

andmutant 74 inHEFcells. The results further

demonstrate the temperature sensitivity of

both mutants in HEF cells and also suggest

that maturation of mutant 74 proceeds at a

slowerratethanthat of wild-typevirus,evenat

33C.

DNA studies. Because of their different

den-sities, HSV-2 DNA (density = 1.73g/cm3) (35)

andcellular DNA(density= 1.70g/cm3)canbe

differentiated by isopycnic bandinginCsCl

so-lution, as shown in Fig. 2. For each profile, the

total amount of [3H]thymidine incorporated

into virusand cellularDNAwascalculated by

summingthe radioactivityinthefractions

com-prising the virus and cell DNA peaks,

respec-tively.

The pattern ofvirus DNA synthesis during

the infectious cycle in HEF cells is shown in

Fig. 3 forwild-type 333 and the two mutants.

Wild-type virus was observed to synthesize

mostofitsDNAduring thefirst9hafter

infec-tion, a findingthat is consistent with earlier

reports (2, 34, 35). Thiswasthecasebothat 33 and 39C, although atthe highertemperature

less virus DNA was made, supporting the

slight temperature sensitivity of HSV-2 in

hamster cells(6).

Virus DNA synthesisin HEF cells infected

by mutant 69 occurred predominantly during

the first 9 h postinfection but, even at 33 C,

mutant 69 directed only half as much viral

DNA synthesis as did the wild-type 333. At

39C the discrepancy between wild-type and

mutant69DNAsynthesisisgreaterthan two-fold, but some mutant virus DNA synthesis

wasobserved.

Theresults obtainedsuggestthat mutant 74

directed relatively little virus DNA synthesis

(comparedwiththat directedbywild-typevirus ormutant69) for the first16h after infection of

HEF cells, although some progeny production

could be detectedatthistime.ThepeakofDNA

synthesis occurred between17 and25h

postin-fection,atboth33and39C. Considerablymore

virusDNAwasmadeatthe lowertemperature,

101

-j 5

x 10

101

0 4 8 12 16 20 24

HOURS POST-INFECTION

FIG. 1. Growth curves of HSV-2 strain 333, mu-tant69, andmutant 74 in HEFcells at 33 and 39 C. Cells were infectedat an input multiplicity of5.0 PFUlcell and were harvested, virus extracted, and

titrated,asdescribed.

but this amounted to only about 30% of the

amountofwild-type virus DNAmadeby HEF

cells infected under similar conditions.The

ob-servationthatmutant 74makesprogenyDNA

"late" intheinfectious cycleisconsistent with

the previous observation that the replication

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FIG. 2. Separation ofvirusand cellular DNA byCsCI equilibrium centrifugation. Thisrepresentative profile shows virus and cell DNAfromacultureofHEF cells that had beeninfectedwith HSV-2 strain 333 17 hpreviouslyand incubated at39C. [3H]TdR wasaddedat 9 hpostinfection, asdescribed. Symbols: x,

radioactivity profile obtainedfrom infected cells; 0, radioactivity profile from mock-infected cell sample labeled with[14C]TdR, whichwascocentrifugedwith materialfrom infectedcells.

cycle of the virus is longer than that ofmutant

69ortheparentalvirusat33 CinHEFcells.

Electron microscope studies. Theprogenyof infection of HEF cells (at 33 and 39C)by

wild-typeHSV-2 333 and bymutants69and 74were

harvested 24 h after infection andwere

exam-ined in the electron microscope. Virus with a

herpes-like morphology was seenin all

prepa-rations andwassubdividedinto particles with orwithoutanelectron-densecore(denoting the presence of virus DNA) and withorwithout a

distinguishableenvelope.

Itsoonbecameapparent, asshown inTable

2, whichrepresentsthe results fromanumber

of experiments, that even at the permissive temperatureprogenyofinfection bymutant69

hada veryhigh ratio ofparticletoinfectivity. It

was earlier indicated (Fig. 1) that 24 h after

infectionat33C theprogenytitersof infections

by wild-type 333 andmutant69werenot

sub-stantially different; thehigh ratio ofparticleto

infectivity indicates that cells infected by

mu-tant69produce alarge number ofvirus

parti-cles, mostof which are notinfectious. Similar

observations weremade when mutant 69 was

grown at 39 C in HEF cells, when ratios of

particletoinfectivityashighas amilliontoone

were notunusual. (The infectivity ofprogeny virus wasalwaysassayedat33CinRKcells.)

Theparticles producedbymutant69-infected

cellswereexamined morecloselytodetermine

whetherthe vastnumber of defective particles

was morphologically abnormal. No difference

wasfound betweenthe ratioofcored(probably

DNA containing) to noncored particles in the

progenyfromwild-typevirus-infected cells and in the progeny from mutant 69-infected cells.

Therewas aslightlylargerproportion of

nonen-veloped particlesin the progenyofmutant 69

infections comparedwiththose of wild-type

in-fections, butthis was notparticularly striking

and was insufficient to explain the defective

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MUTANTS

100

90

80

70

v 60

R0

X 50

e 40 30

201

10

1 9 17 25 1 9 17 25 1 9 17 253341 HOURS POST-INFECTION

FIG. 3. Pattern ofvirus DNA synthesis in HEF

cellsinfected with HSV-2-333,mutant69,ormutant 74andmaintainedat33or39C. Columnsrepresent total [3H]TdRincorporation in theviruspeak (from

CsCl equilibrium density gradient) during 8-h

pulses of labeled material.

TABLz 2. Productionof infectious and noninfectious

progenyby HSV-2 strain333andmutants69and74

inhamstercellsat33and 39Ca

Vi- Growth Particle/in- Nonenve- Nocore/ rus temp(C) fectivityra- loped/en- core

tio veloped

333 33 23-50 10-27 2-10

39 50-100 16-30 8-20

74 33 27-70 88-200 30-70

39 1.5-9x10' 100-300 50-100 69 33 2.3-10x10' 64-100 5-10

39

102-10'

40-200 10-50 6 Virus prepared under different experimental

condi-tionswastitrated in RK cellsat33 C.

(i.e., morphologically normal but

noninfec-tious) nature ofthe greatmajority ofprogeny

produced bymutant69.

Virusproducedin HEFcellsat39Cshoweda

similarpattern.Therewas aslightincrease in

the proportion ofnonenveloped particles com-pared with the progeny of wild-type virus grown at 39C andno significantdifference in the ratio ofcoredtononcored particles. Thus,

there isno obvious morphological explanation

forthe defectivenatureof the bulk of the

prog-enyvirus produced by mutant69.

Progeny produced by mutant 74 at 33 C

ap-pearedtohave onlya veryslightlygreater ratio

ofparticle to infectivity than wild-type virus

progeny, and both the proportion of

nonenvel-oped and the proportion ofemptyparticleswere

somewhatgreaterthaninpreparationsof

wild-type virus. At39 Ctheratio of particle to infec-tivity ofthe progeny was significantly higher

than for wild-type virus, suggesting that

mu-tant 74, like mutant 69, produces "defective" progeny at 39C. Many of the noninfectious progenyofmutant 74appearedto be

nonenvel-oped, and therewas an increase inthe number

of"empty" nucleocapsids producedatthis

tem-perature.

The propensityofmutant 69forproducinga

large number of defective particles was also

demonstratedinpermissivecells. Virus stocks

grown inHELcellsat 33C hadahigherratioof

particletoinfectivity inthecase ofmutant69

thanfor wild-type or mutant 74virus. The

de-gree of defectiveness of virus in stocks of 69

dependeduponthehost cell in which thevirus

wastitrated (Table3).The observations bothat

33 and 39C further support the "host range"

propertiesof thetwo mutants.

Wild-typevirushadessentially thesame

ra-tioofparticletoinfectivity (approximately10to

25particles/PFU) when titratedinRK, HEL,or

BSC-1 cells. InHEFcellsaslightly higherratio

ofparticle toinfectivitywasobserved at39C,

reflectinganapproximately 10-fold decreasein

plating efficiencyof HSV-2atelevated temper-aturesinthese cells. BSC-1 cellsappearedtobe permissivefor all three virusestested, atboth temperatures, and the lowest ratios of particle to infectivity were observed for all viruses in

these cells. RKcells, althoughmorepermissive

than HEF cells formutantvirusgrowth,

partic-ularlyat39C,wereless permissive than HEL

orBSC-1 cells. 3T34E TK- cells supported

lit-tle wild-typeor mutant virusgrowthat39C.

Assay for defective interfering particles.

Since stocks ofmutant 74, and more

particu-larly mutant69, were knowntocontainmany

virusparticles thatwereunabletoreplicateat

39C, a phenomenon that was especially

marked in HEF cells, experiments were

de-signed to determine whether the presence of

mutantviruswould interfere withreplication ofwild-type HSV-2-333 at 39C in HEF cells.

Defectiveinterferingparticleshavebeen

previ-ouslyreportedinHSV stocksprepared byvirus passage at high input

multiplicities

(4) and

also between wild-type MPdk+ and

MPdk-strainsofHSVgrowingin canine cells(33).

EJ -330C

_ -390C

333

69

74

h=

L-

I=1P

no

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TABLE 3. Relative plating efficiency of wild-type andmutantHSV-2 indifferent typesofcellsexpressedas

ratios ofparticle toinfectivitya

Platingefficiency of:

Cells 333wild type ts mutant 69 ts mutant74

33C 39C 33C 39C 33C 39C

HEF 17-25 250-480 200-300 7-9 x 107 40-110 9 x 105

Baby RK 10-18 15-30 100-200 1,500-1,900 20-50 60-100

HEL 17-25 17-25 80-100 750-900 20-50 50-80

BSC-1 cells 9-12 10-18 20-30 100-150 4-10 10-15

3T3-4E TK- cells 30-40 >104 5,000-8,000 >106 90-110 >104

aVirus infectivity was titrated using a plaque assay in which monolayers of cells weremaintained in

medium containing 0.5% methylcellulose for 4 days prior to the counting of virus plaques. Particle counting ofthe stock virus used in each experiment (grown in HEL cells at 33 C) was by electron microscope examination, as described in the text.

Such interference could be demonstrated

(Ta-ble 4). Wild-type and both mutantviruses

se-verely depressed the yield of HSV-2-333 when

the "interfering" virus was added at aninput

multiplicityof 20 PFU/cell. Decreased

concen-tration of interfering virus decreased the reduc-tion in progeny synthesis, but this effect was

least marked whenmutant69wasthe

interfer-ing agent.Itseemsentirelyreasonableto sup-pose that the large number of defective

parti-cles inpreparationsof this viruswas

responsi-ble for interference with normal replication of

HSV-2-333 at39C inHEF. Similar results to

those shown in Table 4were obtainedusing a

constantinput multiplicityof0.2PFU of HSV-2-333 percell and similar multiplicitiesof

"in-terferingvirus."

Interference by mutant defective particles

was also demonstrated using a plaque

reduc-tion technique (Table 5). At 33 C both large

(presumablywildtype) and small(presumably

mutant) plaques were counted, and the total

number of plaques was expressed. At 39C, it

was again observed that mutants 69 and 74

interfered with the development of wild-type

plaques, and again this effectwasmoremarked

forpreparationsofmutant69. Interference

be-tweenviruseswasnotapparentat33C, possi-bly becauseatthistemperatureconditions are

muchmorefavorable for virus growth, which is

hencefavoredoverabortive infection and

inter-ference.

Biochemicaltransformation of3T3-4E TK-cellsbyHSV-2 strain 333 andmutants69and 74.The effect of UV irradiationonthe

infectiv-ityofmutantand wild-type HSV-2 is shown in

Fig. 4. Wild-type strain 333 was found to be

more resistant to UV irradiation than either mutant, with 74 the most sensitive, the same

amount of irradiation causing a 1 to 2 log1o

greaterdrop in titer ofmutant74than of

paren-tal 333.

TABLE 4. Assay for defective interfering particles in stocksofmutantvirus,using virusyield as indicator Input multiplicity of interfering virus "Interfering virus" (PFU/cell)

20 2 0.2

333(Wild type)

Challenge: 2.0 2.0x 105 6.0 x 105 1.0x 106 PFU/cella (20)b (60) (100)

Challenge: 0.2 2.0 x 105 3.0 x 105 1.0x 106

PFU/cellc (20) (30) (100)

Mutant 69

Challenge: 2.0 4.0x 104 2.0x 105 3.0 x105

PFU/cella (4) (20) (30)

Challenge: 0.2 3.4 x 104 5.0x 105 8.0x106

PFU/cellc (3.4) (50) (80) Mutant 74

Challenge: 2.0 1.6 x 106 9.0x 105 1.0x 106

PFU/cella (16) (90) (100)

Challenge: 0.2 1.4 x 105 7.0 x 105 2.0x 106

PFU/cellc (14) (70) (200)

aAll cultures werechallengedwithHSV-2-333wild-type

virus at aninputmultiplicityof 2.0PFU/cell.Control yield =1.0x106fPFU/ml (20PFU/cell). Titers represent the yield of HSV-2(wild-type large plaques). Virus was titrated in RKcells asdescribedinthetext.

bNumbers in parentheses are percentages of control

yield.

eAll cultureswerechallengedwithHSV-2-333wild-type

virus at aninputmultiplicityof0.2PFU/cell.Controlyield = 106PFU/ml(20PFU/cell).Other conditions were similar tothose describedinfootnotea.

3T3-4E TK-cells were exposedto virusthat

had been irradiated for0 to 10 minand, after21

days of incubation in selective HAT medium,

the number ofcolonies that had formedonthe

12 replicate plates for each virus sample was

counted.Ineachexperiment, the reversion

fre-quencyof3T3-4ETK-cellsto a TK+phenotype

was estimated from the number of colonies

formingon12replicate plates containing cells

that had been exposed to UV-irradiated Tris

buffer. Anunfortunate feature of this

transfor-mationassayisthat the reversionfrequency of 3T3-4E TK- cellswas found tobe rather high J. VIROL.

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[image:8.509.276.436.145.598.2]

TABLE 5. Assay for defectiveinterfering particles in mutant stocks using plaque reduction as indicator

Temp No. of

plaques

(C) 10-2 10-3 10-4 10-5 10-6 10-7 333 33 TNTC TNTC TNTC 162 113 96 39 TNTC TNTC 174 164 119 98 69 33 TNTC TNTC TNTC 136 118 112

39 26 40 49 57 85 117

74 33 TNTC TNTC TNTC 142 137 116

39 55 57 61 111 115 125

Aconstant amount of wild-type 333 virus was plated which produced an expected number of plaques=90to 100/ dish at both 33 and 39 C. TNTC, Too numerous to count.

0 us

z

LL

0

(A 0o cr:

0 2 4 6 8 10

MINUTES ULTRA-VIOLET IRRADIATION

FIG. 4. Survival of HSV-2 strain 333 and

mu-tants 69 and 74after exposure to increasing doses of UVlight.Symbols: 0,HSV-2 strain 333(wildtype); 0,mutant69; 0,mutant74.

andvariable; nevertheless, theresultsreported

hereareregularlyreproducible. Reversion

fre-quency wascalculated for each experiment and

subtracted from frequency of foci on

virus-treated plates to give the "transformation

fre-quency."

Unirradiated virus-induced cytopathology

and nocolonieswere seenafterexposureof cells

to unirradiated wild-type virus or mutant 69.

Cells infectedwithunirradiatedmutant 74did

giverise to rarecolonies,buttheseoccurredat

amuchlowerfrequencythan thereversion

fre-quency of 3T34E TK- cells (approximately

1-5) and theirsignificance is obscure.

WithincreasingUVdose,astheinfectivityof

virus preparations decreased, the frequency

withwhich transformedcolonieswereobserved

increased (Fig. 5). The data in Fig. 5have all

been corrected for the mean number of foci

16 14 12 10 8 6 4 w_ 2

-J

0

cc

12

3 0

U 10

U.

0

c 8

z

c4

'u

2 0

10 8 6 4

2 0

0 2 4 6 8 10

MINUTES U/V IRRADIATION

FIG. 5. Biochemical transformation of 3T34E

TK- by HSV-2-333 and mutants 69 and74. Mean numberof foci per plate was corrected for the mean numberof focioncontrol (non-virustreated) plates. The results represent themeanofthreeexperiments, andthe barsgive the standarderrorabout themean.

99

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[image:8.509.54.253.219.491.2]

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observed on control plates. This varied from

0.17to 2.5 indifferent experiments. Significant

transformation was not observed with

wild-type HSV-2-333 until the preparation had

re-ceived at least 2 min of UV irradiation(4.8 x

105ergs/cm2); thereafter, the observed number

oftransformed foci increased. The maximum

frequency oftransformation events (i.e.,

num-bers of foci corrected for meanfoci oncontrol

plates) isapproximately3 x 1O-4.

Mutant 69, which was found tobe more

sensi-tive to UVirradiationthanthe parentalvirus,

induced the formation of significant numbers of transformed foci at lower UV doses than

wild-type 333, and aslightly higher maximum

fre-quency oftransformationwasobserved.

A more striking difference in transforming

ability was shown by mutant 74. This virus, which was the most sensitive to UV

irradia-tion, wasable to transform significant numbers

of cells at alow (12 x 104ergs/cm2) UV dose,

and thefrequencyof transformation after

expo-suretothis virusincreasedrapidlytoaplateau

levelthat was equal to, orslightlyhigherthan,

the maximum transformation frequency

ob-served withwild-typevirusormutant 69.This

plateauwasmaintained for UV dosesfrom2to

10min(4.8 x 105 to2.4 x 106 ergs/cm2). Virus

infectivity droppedtoundetectablelevels after

only5 x 105ergsof irradiation per cm2.

The data presented in Fig. 5 represent the

mean ofthree experiments. Morerecently, two

further experiments, using virus that had been

irradiated for as much as 15min,have provided

Preliminary

characterization of themutants

haddemonstrated that their temperature

sensi-tivity was notduetothermolability ofthe vi-rion(R.W. Koment, unpublished data)ortoa

block in virusadsorptionorpenetrationat39 C

(23). Thiswasconsistent with resultsreported in this communication, demonstrating that a

number ofvirus-directed changes occurred in

HEF cells infected at 39C. Some virus DNA

synthesistook place, host cell DNA

synthesis

was turned off(datanotpresented), andafew

virus particles wereproduced, although these

particlesweremainly noninfectious. The

obser-vationthatmutant69producesmany

morpho-logically normal particles of greatly reduced infectivityis similartothat madebySchafferet al. (37), who reported a C groupts mutantof

HSV-1 that produced apparently

morphologi-cally

normalparticlesofgreatlyreduced

infec-tivity.

Schaffer and co-workers (37) were also ableto

correlate the production of cored and empty

particleswith the DNAphenotype of the virus.

To some extent, similar observations were

made in thisstudy usingmutant 74.This

mu-tant, even atpermissive temperatures in HEF

cells,hadadelayedonsetofDNAsynthesisand

at39 C made very little virus DNA. Particles

produced in cells infected by mutant 74 showed

a preponderance of nonenveloped empty

nu-cleocapsidsat39C.

The "host range" properties of mutants 69

and 74 appearto form a gradualpattern from

fully permissive cells (BSC-1) to extremely

re-strictive cells (HEF). This growth restriction in

different cells isdemonstrated at33 C but, at

39C, it is much more marked. Itseems

reason-able to conclude that the mutants have

diffi-culty in replicating in certain cells and that the

additionalstressofhigh temperature (HSV-2 is

knowntogrowpoorlyatelevated temperatures

[6]) prevents virus growth almost completely.

This conclusion could have importantparallels

invivo, particularly with regardtolatency. It

may be that certain cells ofthe host are

natu-rally resistant to HSV growth or even that

there are certain naturally occurring HSV

strainswhose growth isrestricted in some cells,

e.g., of nervoustissue. Such strain differences

would not be without precedent; Epstein-Barr

virus for example, another herpesvirus of

hu-mans,appearstohave at least twoforms, one of

which canreplicate inlymphocytes,the other of

which cannotreplicate but can transform

hu-mancord bloodlymphocytes (27, 28). It is even

possible, of course, that such strain differences

ofEpstein-Barr virus are due to detectivess,"

likemutants 69 and 74.

The high transforming frequency observed J. VIROL.

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withmutant 74 after relatively low doses of UV

irradiation was a particularly interesting

re-sult in this context. It had been thought possi-ble that mutant 69, which produced a very large number of defective particles, might

prove to be aparticularly efficient transforming

virus. This was not found to be the case, as

extensive cytopathic effects were observed

when this virus was added to cells after low UV doses, suggesting that, although unable to

rep-licate, the majority of mutant 69 particles are

able to induce cell death and, thus, preclude transformation. Mutant 74, although it did not produce a large number of defective particles,

was abnormally sensitive to UV damage. It

seems probable that this property, together

withthe naturally delayed growth cycle of the

virus, tipped the balance toward cell

transfor-mation (rather than lysis). It should be

men-tioned again,however, that there are a number

of shortcomings with the transformation

sys-tem used in this study. The assay was based

strictly on quantitative focus formation, and colonies were not examined for the presence of herpes-specific antigens. Considerable

varia-tion was seen in the number of fociproduced in

different experiments, and the reversion

fre-quency of the 3T3-4E TK- cells was high (1 x

105to 5 x 10-5). Similartransformation

experi-ments using L(TK-) cells (obtained from W.

Munyon) gave transformation frequencies too

low tobe quantified, but again a greater

trans-formingefficiency was observed using mutant

74 than mutant 69 or wild-type 333 virus.

Takentogether, the data on the transforming

potentialof the three virusestestedlead to the

conclusionthat great care must be exercised in

the development ofattenuated herpes simplex

virus vaccines. Mutant 74 is attenuated, both

forgrowth incell culture and for virulence in

mice(22),and on this criterion would be

consid-eredasuitablevaccinecandidate. However,by

selecting forgrowth attenuation, selection has

alsobeen made foraviruswhosecytolytic

prop-ertiesaremorethanusually sensitive to

exoge-nousdamage, but whose transforming

capabili-tiesremain intact. This situation would clearly

be most undesirable inavirusvaccine.

ACKNOWLEDGMENTS

Thiswork was supported by contract no. NOICP53516 within TheVirusCancerProgram of the National Cancer Institute and fellowship IARC/R 622 awarded to Diana Westmoreland fromthe WorldHealthOrganization, Inter-national Agency forResearch on Cancer.

Wethank Ronald Glaser andRoss Farrugia for the elec-tronmicroscopy and itsevaluation and Jenny Kilmer for excellent technical assistance.

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