Host function-dependent induction of defective interfering particles of vesicular stomatitis virus.

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Vol. 25, No. 1 PrintedinU.S.A.

Host

Function-Dependent Induction of Defective

Interfering

Particles of Vesicular Stomatitis Virus

C.YONG KANG*ANDRAE ALLEN

DepartmentofMicrobiology, TheUniversityofTexasHealth ScienceCenter,Southwestern MedicalSchool,

Dallas,

Texas 75235

Receivedfor publication12July1977

Suppression of host cell function bytreatment with actinomycin D prior to

infection

prevented

the induction of defective

interfering

particles

of

vesicular

stomatitisvirus, which had been cloned andpropagatedincellspretreated with

actinomycin D.Replication of defectiveinterfering particles already presentin an

infecting

virus

stock, however,

wasnotaffected

by

pretreatmentofcellswith

actinomycinD.Thus,theinduction, but not the replication, of defective

interfer-ing

particles

appears to be a host cell

function-dependent

phenomenon.

The

implications

of this

phenomenon

for host defense mechanisms

against

virus

infectionsarediscussed.

The existence of defective interfering (DI)

particles

ina

preparation

ofananimal viruswas

described firstby Von Magnus (13) in influenza virus. Huang and Baltimore (6) postulated a

role for DIparticles in host defense mechanisms in

overcoming

theviral infections. The physical and biological properties of DI particles have

been reviewed

previously

(5, 14).

One

of the

major goals

in DIparticle-mediated viral interference research istounderstandthe

mechanism of the induction of DI particles

dur-ing standard virus

replication.

DIparticles are

usually

generated during

serialundiluted

high-multiplicity

passagesofstandard infectiousvirus

(5), but little is known about themechanismof

induction

of DI

particles.

Hollandetal. (4)

sug-gested

that DI

particles

ofvesicular stomatitis virus(VSV)areinducedateachpassageofvirus,

including

thefirstone,and thenareselectively

amplified

during

successivepassages toproduce

a detectable level of DI

particles

in the later

passages.

By using

a

single, freshly

cloned

prep-aration of VSV

(VSVIND),

we found that serial undiluted

high-multiplicity

passages of thevirus

infour different celllinesproduced differentsize

classes ofDIparticlesatdifferentpassage

num-bers

(C.

Y. Kang,T.

Glimp,

J. P. Clewley, and D. H.L. Bishop, Virology,inpress).These data

strongly indicated thatthe induction of DI

par-ticles isdependentupon afunction ofthe host

cells. Accordingly, we have examined the

gen-eration of DI particles ofIndiana serotype of

VSV

(VSVIND)

in cells pretreated with

actino-mycinD. Thisreportdescribestheevidence for

ahostcell function involvedinthe inductionof DIparticles ofVSVIND.

MATERIALS AND METHODS

Cells and viruses.Babyhamsterkidneycellclone 21 (BHK21) was obtained from the American Type CultureCollection;BHKs3was akindgiftof D. H. L.

Bishop, University of Alabama; and B77 strain of avian sarcoma virus transformed rat cells [R(B77)]

wasobtained from Howard M.Temin,McArdle Lab-oratory, UniversityofWisconsin. None of these cell linesproduceanydetectable amounts of endogenous virus.R(B77) cellsweregrown asmonolayer cultures in Dulbecco modified minimum essential medium

(DMEM) containing antibiotics and supplemented with 5%heat-treated fetal calf serum (FCS). BHK21

and

BHKs3I

cells weregrown in the same media

sup-plemented with 10% FCS. All the media, sera, and

antibioticswerepurchasedfrom Grand Island

Biolog-icalCo.,GrandIsland, N.Y. Theprototype strainof the VSVIND used in this paper has been described

previously(2, 8).

Preparation of stock virus from a single plaque isolate. Virus stock ofVSViNDwasobtained fromfive consecutiveplaquepurificationsfollowed by

twoadditionalplaque purificationsincells pretreated

withactinomycinD.Confluentmonolayercultures of eitherR(B77) cellsorBHK21 cellsin 100-mmculture dishes containing approximately 1 x 107 cells were treated with 1

tg

ofactinomycin D perml for24h. Actinomycin D-pretreated monolayer cultures were infected with diluted virusto give approximately 50 plaquesineach culturedish andoverlayedwithagar. Single, wellisolatedplaqueswerepickedandusedto inoculate other cultures alsopretreated with

actino-mycinD.The virusesfromthis secondplaqueisolation fromeitherR(B77)cellsorBHK21cellswereusedto infect cellspretreatedwith1ygofactinomycinD per mlat amultiplicity ofinfection of0.1 PFUpercell. Viruswas grown for 16 h, at which time the virus fluidwasharvested andcentrifugedat600xgfor15 min to remove thecellular debris. Portions of virus 202

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VSV DEFECTIVE INTERFERING PARTICLES

fluid (3 ml) were stored at -75°C. The titer of the stock viruswasapproximately2x 109PFU/ml.

Successiveundiluted passages ofVSV,D. Cul-ture dishes (100 mm) containing approximately 1 x 107cells in monolayer were treated with 1

iLg

of acti-nomycin D per ml for24h. Aparallel control culture was nottreated with actinomycin D. The monolayer cultureswereinfected with0.5ml of the stock virus to give a multiplicity of infection ofapproximately 100PFU percell for the first passage; for the subse-quent passages, 0.5ml per 100-mm dishof undiluted virusfrom theprevious passage was used. The virus wasadsorbedto thecellsfor45min at37°C, and 7 ml of DMEMsupplementedwith5%FCSwasadded followed by incubation at 37°C for 7 h in a CO2

incubator with saturated humidity. The virus was harvested and centrifuged at 600x gfor 15 min to removecellular debris, and the viruswasanalyzed by plaque assay on BHK53 cellmonolayers in a 60-mm culture dishand/or by sucrose gradient centrifugation asdescribedpreviously (8, 9).

RESULTS

Serial undiluted passages of

VSVID

in cellswith and without

actinomycin

D pre-treatment. Serial undiluted

high-multiplicity

passagesofa

single

clone of

VSVIND

in four

cell

linesproduceddifferent size classes of DI parti-cles atdifferent passage numbers

(Kang

et al., inpress). These data indicate that the induction ofDIparticlesisdependenton ahost cell func-tion. This

prompted

usto

investigate

induction ofDI

particles

in cells pretreated with

actino-mycin D,

aknown inhibitor for DNA-directed information flow (12) withnoknowneffects on

VSV

replication

(9). The stock viruswasplaque

purified

and

prepared

in cells

pretreated

with

actinomycin

D as described

above,

and serial

undiluted

passageswerecarriedoutincellswith and withoutactinomycin D pretreatment. Fig.

la shows the titer of VSVIND propagated in R(B77) cells with and without actinomycin D

pretreatment. The untreated R(B77) cells

yielded

approximately

thesameamountof virus

up topassage4and thenshoweda

rapid

reduc-tion in virus titer

during

the subsequent pas-sages. In contrast, when the same virus was

propagated

in R(B77) cells pretreated with

ac-tinomycin D,

the titer of VSV in eachpassage uptopassage11remainedconstant.The number of cells in

actinomycin D-pretreated

cells was lessthan that ofcontrol culturesatthe time of infection; therefore, virus titers in actinomycin D-pretreatedcells are lower than in control cells. WecarriedoutsimilarexperimentswithBHK21 cellstoruleoutthe

possibility

that the

phenom-enonis

dependent

upon a

particular

cell type. There was no reduction in virus titer up to passage 10 when the virus was propagated in

BHK21 cells pretreated with actinomycin D,

E

D

U-

0-Number of undiluted passages

FIG. 1. Yieldof standard infectious virions from cells with and without actinomycin D pretreatment during successive undiluted passages. The initial stock viruswasprepared by the consecutiveplaque isolation method described in the text. Confluent

monolayerculturescontaining approximately1x107 cells per100-mmculture dish werepretreated with 1 ,ug of actinomycin D per ml for24 h (Act. D). The celldensity in theparallelcontrol cultureswas

ad-justedtogiveabout1 x 167 cells per100-mmculture dish. Theconfluentmonolayer cultureswereinfected with 1 x 109 PFUof the stock virusfor the first

passage. Thesubsequentpassagesweremadeby

in-oculating1x107cells perculture dish with 0.5 mlof undilutedpreviouspassage. At the endofeach 7-h passage, theyieldof infectious viruswasmeasured

byplaqueassayon aBHK53cellmonolayerina 60-mmculture dish.Actinomycin D-pretreatedcells(Act.

D)and the cell typesareindicated.

whereas it took6passagesto

give

approximately

a3-log reduction from the

original

titerin the absence of

actinomycin

D

(Fig.

lb).

The

rising

titer ofVSV atpassage 8 in

R(B77)-untreated

cells

(Fig. la)

andpassage 7 in

BHK21-untreated

cells

(Fig.

lb)

maydemonstrate the

cyclic

pro-duction

ofinfectious B

virions,

as has been

re-ported (10).

To determine whether the reduction of titer after serial undiluted passages of VSV was the result of the

generation

of DI

particles,

the

vi-ruses from each passage were

analyzed

by

su-crose

gradient

centrifugation. Figure

2

demon-strates thatatleast three different size classes

of DI

particles

were

produced

in

R(B77)

cells whentheviruswas

propagated

incells without

actinomycin

Dpretreatment,whereasno

detect-able amounts of DI

particles

were

produced

from the cells

pretreated

with

actinomycin

D. Two differentsize classes of DI were

produced

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FIG.

2. Sucrosegradient analysis of total virus particles from successive passages. Samples oflysates

fromfour culture dishes of each passage illustrated in Fig. 1 were centrifuged at 600 x g for 15min. The virusparticles werepelletedfrom thesupernatant bycentrifugation at81,000 xgfor 75min ina Spinco SW27 rotor, suspended in 0.3ml of phosphate-buffered saline, and layeredon a linear5 to 30%sucrose gradient made inphosphate-buffered saline. After centrifugationat110,000xgat5°C for35min in aSpinco SW41 rotor, thegradients werephotographed. Thearrowsindicate the standardinfectious particles. The three additional bands above thestandard bandshown in passage6inR(B77) cells and thetwoadditional bands above the standard virion band shown inpassage 5 in BHK21 cells are DIparticles. Electron microscopic examinationsofmaterial in bandssedimenting just below thestandardvirion revealed only

clumpsofparticlesidenticaltostandard B virion. Theclumpedparticleswereinfectious.

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VSV DEFECTIVE INTERFERING PARTICLES

in untreated BHK21 cells, and no DI particles weredetectable when the viruswaspropagated

inactinomycin D-pretreatedcells(Fig. 2).

We haveexamined thestate ofthecellsafter

24h oftreatmentwithactinomycin D by deter-mining the cellular RNA synthesis. Table 1

showsthe[3H]uridineincorporation into R(B77) andBHK21 cellsaftertreatmentwiththree dif-ferent concentrations ofactinomycin D. Approx-imately 95% of cellular RNA synthesiswas

in-hibitedby1,ug of actinomycin Dtreatmentper

ml for24 h.Consequently,mostcellular

macro-molecularsynthesesmusthave beendepressed

atthe time of virus infection, as shown

previ-ously (9).

Production of DI particles in cells with

and without actinomycin D pretreatment using stock virus knowntocontain DI par-ticles. We examined thegrowth of DI particles incellspretreated with actinomycin D byusing

virus stocks containing known DI particles to

determine whether theproduction of DI parti-cles is alsodependent upon acellular function.

Two stock viruses containing DI particles

pre-pared from cells without actinomycin D

pre-treatment (that is, the passage 5 virus from R(B77)cellsandthepassage4virus fromBHK2,

cells shown in Fig. 2) were propagated in cells

pretreated with actinomycin D and in untreated

cells. The data inFig. 3 demonstrate virtually

identical patterns of DI particle production in cells with or without actinomycin D pretreat-ment.The actinomycin D-pretreated cells

pro-duce DI particles and the standard infectious virions (Fig. 3, BHK21 passage no. 2) as

effec-tivelyasuntreatedcells,demonstratingthat

ac-tinomycinD-pretreatedcells arecapable of

sup-porting the growth of not only the standard infectious virionsbut also DI particles already present in a virus stock. Accordingly, we

con-clude that the induction of DIparticle is host function dependent, but their production does

notrequire thesamehostfunction.

DISCUSSION

Although indirect evidence suggested that host function(s) determine the generationof DI

particles,we knowverylittleaboutDI particle induction during serial undiluted passages of standard infectious virions. It has been postu-lated that DIparticles ariseasartifacts in viral

preparationsorby mutationaleventsduring the

replication of standard virions (3, 13). However, Hollandetal. (4) havenotbeen abletogenerate

DI particles by mechanical disruption or UV

irradiation of the virus.

We have demonstrated in thispaperthat the

induction ofDIparticles requiresafunction of

host cells that is sensitive to actinomycin D, sincewecouldnotinduceDIparticles when the viruswasplaquepurified and propagated in cells

pretreated with actinomycin D. It is important

tostress thepoint that onemustplaque purify

the virus in cells pretreated with actinomycin D to demonstrate the lack of induction of DI

particles. We have performed the above

experi-mentwithplaque-purified stock virus prepared in the cells without prior actinomycinD treat-mentand found that DIparticleswereinduced

at later passages even though the virus was

propagated in actinomycin D-pretreated cells. Ourresult is consistent with the idea of Holland

et al. (4) that DI particles are generated from

the first passage and amplified during subse-quentpassages.Our data suggestthatinduction of DIparticles by virus-specified premature

ter-mination of RNAreplication, improper cleavage

of messenger RNA transcripts, or mutational

eventsofthe virus itselfduringthe virus repli-cationareunlikely. Wecannotruleoutthe pos-sibility that there are virus-induced cellular

function(s) thatmayberesponsibleforinduction

of DIparticles.

Our results define another possible host

de-fensemechanismoccurringatthe cellular level that protects the host from a highly cytolytic

TABLE 1. RNAsynthesisin cellspretreated with actinomycin D for24ha

Concnofactinomycin R(B77)cells

BHK21

cells

D

[:H]uridine

incorpo- Residual RNA

['H]uridine

incorpo- Residual RNA (ug/mlofmedia) rated (cpm) synthesis(%) rated (cpm) synthesis (%)

0 610.700 100.0 533.500 100.0

0.5 37.810 6.2 32.780 6.1

1.0 30.040 4.9 28.200 5.2

2.0 20.680 3.4 22.160 4.2

aApproximately

1 x 107

cells

per100-mmculturedish wereincubated with 10mlof DMEMsupplemented

with5%FCS and theindicatedamountsofactinomycinDfor24h. TheactinomycinD-pretreatedcellswere labeledwith 5yCiof[3H]uridine(21Ci/mmol, Schwarz/Mann) per culture dishfor 7 h. Trichloroacetic

acid-precipitable radioactivity in the cells was determined by the method described previously (9). Countsper minuteof[3H]uridine incorporationrepresentthe total RNAsynthesis in1.5x 10"cells. Thevaluesarethe average ofdeterminations fromduplicateculture dishes.

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FIG. 3. Production ofDIparticles in cells

pre-treated withactinomycin D.Approximately 1 x 107 cellsper100-mm culture dishwerepretreated with1

jigof actinomycinD(Act.D)permlfor24h,orleft

untreated(control),andinfectedwith the virusfrom

passage5ofthe controlR(B77)cells(Fig. 2)and the virusesfrompassages 1and4ofthe controlBHK21 cells(Fig. 2).Viruswasgrownfor7h andanalyzed in sucrosegradients as describedin the legend to Fig.2.

virusinfection. DIparticles mayplay acritical

role in host defense duringthe initialinfection

ofvirus, especially with fast-growing cytolytic viruses. In such cases, the host is unable to respondquickly enoughtocombat theinfecting

virusearlyininfection withproductionof

pro-tectinglevels ofantiviralantibodies. Other

pos-sible host defense mechanisms, such as

induc-tion of interferon (7) or thegeneration of cyto-toxic T cells(15), maynotbe as effective early ininfectionasDIparticle-mediated viral inter-ference. We have evidence thathost cell function is critical for the induction of DI particles and suggest that the induction ofthese particles may not be a self-destructive viral mechanism, but rather a host cell function-dependent

mecha-nism that will protect the host from further spread of virus.

ACKNOWLEDGMENTS

We thank B. Ozanne, J.Bednarz-Prashad, and J. Shadduck for their detailed reviews ofthis manuscript. We are very grateful to T. H. Stoudt,MerckSharpand Dohme forthe generousgiftofactinomycinD.

Thisinvestigation was supported byPublicHealthService grantsCA 16479 and CA 20012 from the National Cancer Institute.

LITERATURE CITED

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10. Palma, E. L., and A.Huang.1974.Cyclicproduction of vesicular stomatitis virus causedby defective interfering particles.J. Infect. Dis. 129:402-410.

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