Viruses isolated from cells persistently infected with vesicular stomatitis virus show altered interactions with defective interfering particles.

0022-538X/80/11-0627/05$02.00/0

Viruses Isolated from

Cells

Persistently Infected with

Vesicular Stomatitis Virus Show Altered Interactions with

Defective Interfering Particles

FRANK

M.HORODYSKIAND JOHN J. HOLLAND*

Department of Biology, University ofCalifornia,San Diego, La Jolla,California92093

Virusmutantsisolated

from

persistent infections ofvesicular stomatitis virus

in BHK-21 cells were much less susceptible to interference mediated by the

defective interfering particle used to establish the persistent infection. This

mutational change

occurred as

early

as 34

days

in the persistent infection and continued forover 5 years.Theearliest variants showedno

oligonucleotide

map

changes and no difference in the temperature-sensitive phenotype from the

original

virus, but the later variants exhibited extensive map changes. These

resultssuggest apossiblerole for defectiveinterfering particles in the selection of

themutants.

A number of animal viruses which are

nor-mally cytocidal are capable of establishing a

long-term persistentinfection in culturedcells.

Although the precise mechanisms responsible

for the establishment and maintenance of

per-sistent infections of RNA viruses havenotbeen

clearly defined,

anumber offactors have been implicated: (i)

temperature-sensitive

mutants

(18, 27),

(ii)

interferon

production (14,

15,

19),

and

(iii)

defectiveinterfering (DI)particles (1,5, 10, 17, 20, 22,26).Each of these factorsmayplay

agreater orlesser role

depending

onthe virus-cellsystemstudied.

Huang and Baltimore

(7)

first

suggested

that DI

particles might play

an

important

role in viral

persistence.

The

best-characterized

persist-entinfection in which DI particles playamajor role is that of vesicular stomatitis virus

(VSV)

inBHK-21 cells (5, 6). DI

particles

were neces-sary for both the establishment and

mainte-nanceof the

persistent

infection

(5).

The

BHK-21CAR4

persistently

infectedcellline has been maintained for 6.5 yearsandcontinues toshed low levels of standard virus and a

constantly

changing population

of DI

particles.

Standard virus isolated from the

persistent

infection at various timeswas

analyzed by

T1 oligonucleotide

mapping

ofthe viral

RNA,

and it was shown that thevirusshed

by

thisculture is

undergoing

extensiveand continuous mutational

change (4).

Mutationsareexhibitedinall five viralproteins andinthe endsoftheviralRNA

(21,

23).

The recovered virues exhibita

small-plaque

pheno-type; they have become

increasingly

less

cyto-pathic

and canestablish a

persistent

infection after69months withoutadded DI

particles (6,

21). Little is known about the selective

mecha-nisms involved inpromoting sucha rapid

mu-tationrate,and it isdifficulttodetermine which mutations havebiological importance since the mutational evolution of the virus is both

exten-sive and continuous.

Many investigators have obtained mutations inthe virusisolated from

persistent

infections in othersystems(1,12, 13, 18, 24,25).These viruses often exhibitasmall-plaqueor

temperature-sen-sitive

phenotype.

Of

particular

interestwasthe

finding by Kawai and Matsumoto (9) that a

small-plaque

variant ofrabies virus isolated from

a

persistent

infection was less

susceptible

to interference

by

the

original

DI

particle

used to establish this persistent infection. They

pro-posed

that this

small-plaque

variant could be

effectively

selected since it is much less

suscep-tibleto interference

by

the

preexisting

DI

par-ticles.

Wereporthereasimilar

finding

in thecaseof

persistent

infections ofVSV in BHK-21 cells and

present a quantitative preliminary characteri-zation of these viruses.

The persistently infected

line

studied here

(designatedCAR4)was

originally

establishedby

coinfection of BHK-21 cells with ahigh

multi-plicity

of the cloned tsG31 mutant of the VSV Indianaserotypeand itshomologousDIparticle (5). After establishment of thepersistent

infec-tion at

330C,

thecellsweremaintained at

370C.

Virus was isolated at various

times

from the

persistently infected culture by cocultivationof carriercells and nornalBHK-21cells.Virus was

plaque purified,andhigh-titerclonalpoolswere

prepared forusein thesubsequent

experiments.

Wethenexaminedthe

susceptibility

ofthese virusestointerferencemediatedbythe original

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628 NOTES

DI particle used toestablish this persistent in-fection (tsG31 DI). We also quantitated the abil-ity of the recovered virusestoreplicate this DI particle. Thiswasdonebycoinfecting identical

monolayers of BHK-21cellswith theinfectious

virus at a multiplicity of5 PFU/cell together

with increasing amounts of the purified tsG31 DI particle. Interference was quantitated by

plaqueassayof the yield. Replication of the DI

particle was examined after velocity gradient

centrifugation ofprogenyvirus onsucrose

gra-dientstoseparate standardvirus and DI parti-cles. Wequantitated theDIparticleyield

spec-trophotometricallyasdescribed in the legendto

Fig.1.Atypicalsucrosegradient profile is shown

in Fig. la. The resultsareshown inFig. lb and

candsummarized in Table1.Themixed

popu-lation of virusisolated from the persistent infec-tionat 75days contained 70%smallplaques (1

to2mm). The remainder of the plaqueswereof

asize comparable to the tsG3M standard virus (4 mm). The small-plaque phenotypebredtrue,

and no detectable interference or DI particle replicationwasobservedevenatalevelofinput

DIparticles which givesover1 log interference with the homologous standard virus. The large-plaque virus at 75 days postinfection (75-day LP)wasstill subjecttotsG31 DI particle-medi-ated interference and still replicated that DI particle, althoughsomequantitative differences wereobserved. At 14months, the virus isolated

from thepersistent infectionwasoflarge-plaque phenotype andwasonceagain sensitivetotsG31 DI particle-mediated interference. In contrast, the small-plaque virus isolated at 5 years was

insensitive tothese levels of the tsG31 DI

par-ticle. For each virusexamined, the amount of interferenceobservedwasdirectlyrelatedtothe level of DIparticlereplication (compare Fig. lb andc). This correlationshowsthat eitherassay canbequantitatively usedtostudyDI

particle-mediatedinterference.

To determine how quickly this mutational changecan occur in the standard virus during

persistent infection,aseparatecarrier line (des-ignated CAR21)wasestablished in BHK-21cells

by the method by which the CAR4 persistent

infection had been established, and with the exception that these carrier cells were

main-tainedat33°Catalltimes (5). Viruswasisolated

by direct plaquepurification from the

superna-tantof thepersistentinfection. Figure ld ande

shows thatthis mutationwhichconfersa

rela-tive resistance to the original DI particle can

take place by 34 days,atwhichtime asimilar

mixed population of small-plaque (34-day SP)

andlarge-plaque (34-day LP) virus was found.

The34-daySP viruswasnotsensitiveto

inter-ference mediated by the tsG31 DI particles at

the levels of input DI particles employedin this

experiment. However, the virus isolated at 24

days in this persistent infection was entirely

largeplaqueandshowed normal levels of

inter-ferencebythe tsG31 DIparticle (Table 1). Wenextdeterminedwhetherextremelyhigh multiplicitiesofthe tsG31 DIparticlecould in-terfere well with the 34-day SP and 75-day SP viruses. Detectable levels of DI particle

replica-tion and interference did occur with these

vi-ruses at aninputof 10 DI units per culture, and greater than 90% interference was obtained

when 100 DI units of the tsG3M particle were

used. However, weobservedthat thetsG31 DI

particle always depressed theyield of homolo-gous tsG31 virusat least 10- to1,000-foldgreater

than it depressedtheyieldof34-daySP and

75-day SPviruses,evenunder the extreme interfer-enceconditions ofhigh inputDIparticles.

Sim-ilar results were seen if a low multiplicity of

infection (0.01 PFU/cell) of standard virus was

employed with a high inputoftsG31DI parti-cles,and the virusyieldwasassayed duringthe first cycle of infection (at 9 h postinfection). Clearly the mutant viruses are much more

re-sistant tothe

original

DI

particle,

but this

re-sistanceis

quantitative

andnotabsolute. Wenextcarriedoutreciprocal experimentsto

determine whether the originalstandard virus

(tsG31)

is

subject

tointerference

by

DI

particles

generated

by

the virus variants isolated after various periods of persistent infection. BHK-21 cellswerecoinfected withahigh multiplicity (5

PFU/cell)

of standardvirusand5to 10DIunits of each DIparticlestudied. Table 1showsthat

greater than 90% interference occurred with the

original

tsG31 standard virus

production

when

5 to 10 DI units of either the 34-day SP DI

particle

orthe 5-year DI

particle

wasused. It is evident fromthese results that this mutational change is unidirectional. Standard virus

under-went amutationalchange which rendered it less sensitivetothe

original

DI

particle,

whereas DI

particles

generated

by these viruses remained

potent interfering agents against the original

standardvirus.

Itwasofinteresttodetermine whether these mutations whichconferresistance to the tsG31 DIparticle alwayscause a detectableT1 oligo-nucleotide map change in viral RNA. Cloned viruswaslabeled with32Pandpurified. The viral RNAwasisolatedand digested with

T,

nuclease,

and theresulting oligonucleotideswere mapped ontwo-dimensionalgels as previously described

(4). The oligonucleotide mapsof the large- and

small-plaque viruses isolated at 75 days from

CAR4 and34daysfromCAR21areidenticalto

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2 DI STD a

0

TOP BOTTOM

b

O.OI Li2348 55

4

:8' ~~~~~~~~~~~~3

DI UNITS/CULTURE DI UNITS/CULTURE

>

d

in

e

>0.1 cm

2

0.01'~~~~~~~~~~~~~~~~~~~

I 2 3 4 3 2 3 4 5

DI UNITS/CULTURE DIUNITS/CULTURE

FIG. 1. Interference and replication properties of thetsG31 DI particle when coinfected with standard viruses isolated from CAR4 (b and c) andCAR21(d and e). Matched monolayers of 4 x 107BHK-21 cells were coinfected with cloned standard virus at a multiplicity of infection of 5PFU/cell andtsG31 DI particles which were purified by two successive centrifugations in a 5 to 40%(wt/vol) sucrosegradient. Since these

virusesare temperature sensitive, allinfections and assayswere done at33°C. This DI particle purification procedure reduced the level of contaminating standardvirusto less than 1 particle per 108(3).Culture fluids wereharvested at 48 h postinfection and assayed for infectivity by plaque assay. Progeny virus was pelleted and centrifuged on a 5 to 40%(wt/vol) sucrose gradient (3). The gradients were fractionatedcontinuously

fromthe top through an ISCO model UA-5 absorbance monitor which measures absorbance at 254 nm, and the DI particle yields werequantitated by weighing the peaks. Atypicalsucrose gradient is shown in (a). V, PFU per milliliter after additionofvarious doses of DIparticles; VoPFUper milliliter without addition of DI particles. (b andc)@*,tsG31;E475-daySP;E475-day LP;*,14 month;0,55year.(d and

e)@*,

tsG31;

4l

34-daySP;EN, 34-day LP.

NOTES 629

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[image:4.499.60.253.75.279.2]

630 NOTES

TABLE 1. Summary of interference properties of standard virus andDIparticles isolatedfrom

persistentinfectiona

Inter-Cell line Standard DIparticle (90%or

greater)

CAR4 tsG31 tsG31 +

75-day SP tsG31

75-day LP tsG31 +

14 month tsG31 +

5year tsG31

tsG31 5yr +

5yr 5yr +

CAR21 tsG31 tsG31 +

24day tsG31 +

34-day SP tsG31

34-day LP tsG31 +

tsG31 34-day SP +

34-day SP 34-day SP +

34-day LP 34-daySP +

aMonolayers of BHK-21

cells

werecoinfected with

standard virus at amultiplicity of infection of 5 PFU/ cell and 5 to 10 DI units ofpurified DI particles. One DI unit isdefined as the number of DI particles in the inoculum which gave 37% of theyield of DI particle-free inocula (i.e., which gave one hit biologically [2]).

the

original

tsG31virus

(data

not

shown).

It is not surprising that these mutations are unde-tectable by

T,

oligonucleotide mapping

of the viral RNA, sinceonly about 10% ofallmutations result in alterations in the unique oligonucleo-tides.

We next determined whether the tempera-ture-sensitive

phenotype

of the recovered

vi-ruseshad beenalteredduring thecourseof the persistent infection. The

yield

of these viruses

grown at33,37,and

390C

wasdetermined

(Table

2). Allof the viruses recovered from the

persist-entinfectionwerestilltemperaturesensitiveas evidencedbyatleast a

2-log

reductionin

yield

when the viruses were grown at 390C as com-pared with the yield when the viruses were grown at

330C.

There are undoubtedly many selective pres-sures exerted on the virus during a persistent

infection,and DIparticlesappear toprovideone type ofselective pressure. The virus, once

se-lected for greater resistance tothe original DI

particlepopulation can then generate DI parti-cles which can interfere withthereplication of thenewly selected virus as well as theoriginal

viruses

still

present inthe population. The

pop-ulation ofDI particlespresent has been shown to change during the course of the persistent infection(5).We do not yetknowwhethernewly arising DIparticlesinpersistent infections can

select for multiple rounds of mutations in a

similar manner or whether this selection only

occurs once, soonafter the establishment of the

persistent infection. Wearecarryingoutfurther

isolation of viruses fromCAR4andCAR21 and testing their susceptibilitytointerference

medi-ated by these DI particlestoresolve this

ques-tion.

We haverecently obtainedsomeevidence that

DIparticles might be involved in the selection ofmutations. We repeatedly passaged standard virus in BHK-21cellswithout dilution(toensure

thathigh levels of DI particlesarepresentduring

each serial passage). These serial undiluted

pas-sages appeartobegenerating mutations detect-able by

T,

oligonucleotide fingerprinting at a

much faster rate than passage either at high dilutionoralternating high and low dilutions (4,

21; K.Spindler, unpublished data). DI particles

may be partly involved directly by selection

effects and perhaps indirectly by slowing virus replication rates and prolonging cell survival times.However,manyother factors in

high-mul-tiplicity passages (such as complementation

amongmultiplemutantvirusesinthesamecell)

mayplayarole.J. S.Youngner, E.V.Jones,M.

Kelley, and D. W. Frielle (submittedfor publi-cation) have observedastriking increase in the

percentageoftemperature-sensitive mutantsof VSVpresentin thevirus population after 4 to

10 high-multiplicity passages in L cells.

How-ever,they didnotobserve this in BHK-21 cells

unlesstheypretreated the cells with actinomy-cinD, and they couldnotcorrelate the increase intemperature-sensitive mutantswith DI

par-ticle production. They postulate the

involve-mentof hostcellfidelity factors. Further work will be needed to assess the influence of DI

particlesonaccumulation ofmutations in virus

populations.

The location of the sites of mutationonthe

viral genomewhich conferarelative resistance

TABLE 2. Growth characteristicsof viruses isolated from persistent infectiona

Yieldat Yieldat

390C/

Virus 370C/yield at yieldat3300

330C elat3C

ts31 0.24 0.00025

34-day SP 1.0 0.0093

34-dayLP 0.04 0.00025

75-day SP 0.50 0.0014

75-day LP 0.84 0.010

MSB(wild type) 0.69 0.11

aMonolayers of BHK-21

cells

were infected at a

multiplicity of infection of 5 PFU/cell. Infections were carriedout at33, 37, and 39°C.Virusyield was deter-minedby plaque assay at 33°C.

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toDI particles shouldshedsomelight onthose

regions of the viral RNA which play a role in

autointerference. It has been suggested that the

complementary terminal sequences present in

the DI particle RNA may contribute to the

replicativeadvantage of the DI RNAduringan

infection (8, 16). Since the 5' end of the viral

RNAis conserved in theRNAof the DIparticle

(11, 16), and itscomplement is substitutedatthe 3' end, both of these sequences would be

re-flected in the 5' end of the viral RNA. It has

been shown that the 5' end of the viral RNA

mutatesmuchmorerapidly than does the 3' end

during persistence (21, 23). The virus variants described here may result from a coordinated

coselection of mutations in the 5' endof theviral RNAand in the gene(s) coding for thereplicase

(andpossiblyothergeneproducts).Sequencing

andhybridization studiesto testtheimportance of theseregions of the genome arebeing

initi-ated.

We thank EstelleBussey forexcellenttechnicalassistance. We also thank Julius Youngnerforcommunicating hisresults

tousbeforepublication.

This workwassupported byPublicHealthServicegrant

Al14627fromthe National Institutes ofHealth.F.M.H.was

supported byPublic Health ServicePredoctoral Traineeship GM07313.

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