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Inhibition of pseudorabies virus replication by vesicular stomicles virus I. Activity of infectious and inactivated B particles.

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JOURNAL OF VIROLOGY, May 1976, p. 526-533

Copyright©1976 AmericanSocietyforMicrobiology PrintedVol.in18,U.SA.No. 2

Inhibition

of

Pseudorabies

Virus

Replication

by Vesicular

Stomatitis Virus

I.

Activity of

Infectious and Inactivated

B

Particles

EDWARD J. DUBOVI' ANDJULIUS S. YOUNGNER*

Department ofMicrobiology, School of Medicine, University of Pittsburgh, Pittsburgh,Pennsylvania 15261

Receivedfor publication5December 1975

Infectious B particles of vesicular stomatitis virus (VSV) are capable of

inhibiting the replication ofpseudorabies virus (PSR)in avarietyofcelllines.

Even under conditions of an abortive infection in acontinuous line of rabbit corneacells (RC-60),Bparticlesinterfere with thereplicationof PSR withhigh

efficiency. Particle per cell dose-response analysis of B particle populations

revealed that thenumber of VSV particlescapableofinhibitingPSRreplication exceeds the number of PFUbyafactorof32 to 64. WhenBparticlesaretreated

withUVirradiation,adrastic increaseinthemultiplicityofinfection is required

toinhibitPSRreplication. Whereasoneinfective Bparticlepercell issufficient to preventreplicationofPSR, 800 to1,000VSVparticlesrendered noninfective

by UV irftdiation are required to compensate for the loss ofVSV synthetic

activity that results fromirradiation.Temperature-sensitivemutants

represent-ing five complementation groups ofVSV were tested at lowmultiplicities of

infectionfor their effectonPSR replication atthe nonpermissive temperature.

Generally,theabilityofthedifferentcomplementationgroups to

amplify

virion

products at the nonpermissive temperature is associated with their abilityto

inhibit PSR replication. Theseresultsimply thatatlowmultiplicities of infec-tion, amplification ofinfecting VSVcomponents is necessaryfor inhibition of

PSR replication, but at high multiplicities of infection with VSV, a virion

component can preventPSRreplicationinthe absence of denovoVSV RNAor proteinsynthesis.

Youngneretal. (20) reported that vesicular stomatitis virus(VSV)completelyinhibitedthe

replication of pseudorabies virus (PSR) in a

continuous line of rabbitkidney cells (RK-13). Rabbit cellsincultureareunusualinthat

pre-treatment of such cells with interferonfailsto

inhibitthe replication of DNA viruses,whereas inhibition of RNA viruses is normal (20). Therefore,inRK-13cellspretreated with

inter-feron, VSVwasunabletosuppressthe

replica-tionof PSR. This suggested that theinhibition

of PSRreplication of VSViscausedby a

prod-uctsynthesizedduringthe VSVreplication

cy-cle. The present study reports the conditions necessary for inhibition ofPSRreplication by VSV in a variety of cell lines. VSV rendered noninfective by UV irradiation and tempera-ture-sensitive (ts) mutants representing five complementationgroupsof VSVweretested for theirabilitytoinhibit the replication of PSR.

1 Present address:Department ofMicrobiology,School of Medicine, University of Virginia, Charlottesville, Va.

22901.

MATERIALS AND METHODS

Cells. Primary chicken embryo (CE) cells,mouse L cells (clone 929),aline (BHK-21) of hamster kid-neycells, and a line (MDCK) of canine kidney cells were propagated in Eagle minimal essential

me-diumplus4%calf serum. Cell lines of rabbitkidney

cells(RK-13),rabbit cornea cells(RC-60),and

mon-keykidney cells (BSC-1) were grown in MEM plus

10%fetal calfserum.

Viruses. The large-plaquemutantof VSVIND (L, VSV) described by Wertz and Youngner (16) was grown in BHK-21 cells.Monolayersin32-ounce(ca. 960-ml) culture bottles were infected with less than

0.01PFU per cell to avoid theproduction of defective interfering (DI) particles. Analysis of [3H]uridine-labeled viral particles by sucrose gradients failedto

detectDIparticlesinlysatesproduced under these conditions. A single pool of L, VSV was used throughout this study. Virus for this pool was con-centratedbypolyethylene glycol 6000 as described

byMcSharry and Benzinger (14) and was partially purified bypelleting the virus through a 50% glyc-erol cushion. Virus prepared in this manner was stored at -70 C.Temperature-sensitivemutantsof

VSVIND were obtained from R. R. Wagner. Well-526

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isolated plaques from terminal dilutions incubated at 32C wereused to produce pools of each mutant in BHK-21 cells at 32 C. Theefficiency of plating (39.5/ 32) in CE cellswas less than 10-4 for all clones used inthis study. PSRoriginally obtained from Robert Sydiskis was grown in RK-13 cells and assayed in CE cells.

Chemicals. [3H]uridine (specific activity, 25 Ci/

mmol) was purchased from New England Nuclear Corp., Boston, Mass.Cycloheximidewaspurchased

fromUpjohn Co., Kalamazoo, Mich., and actinomy-cin D wasobtained through the courtesy of H. B. Woodruff of Merck, Sharpe andDohme.

Double infections with VSV and PSR. Unless otherwisestated, VSV and PSR were added simulta-neously to cell cultures in 60-mm petri dishes at an inputmultiplicity of infection (MOT) of 5 to 10 for each virus. After an adsorptionperiodof2h at 37C inavolume of 1.0 ml, theinoculum wasremoved by three washes with Eagle minimal essential me-dium. Three milliliters of growth medium was added, and the cultureswereincubated for 18 to 20 h

at 37C. Fluid plus cells were harvested, and the suspension was sonicated torelease cell-associated

virus. To titratethe PSR, it was necessary to neu-tralize the infectivity of VSV. This was accom-plished by adding anti-VSV antibody to the 10-2 dilution of the virussample and incubating at37 C for 1 h. The few VSV plaques that occasionally survived this treatment could easily be discrim-inated from PSR plaques.

In vivo assay of primary transcription by the virion-bound polymerase of VSV. The in vivo assay of primary transcription by the virion-bound polym-eraseof VSV was done accordingtoMandersetal. (10).UV-irradiated orunirradiated VSVwasadded

tomonolayers of BHK-21 or RC-60 cellsatanMOI of 25 (calculatedfromtheunirradiated sample)for1h

at 4C. Afteradsorption,eachmonolayer,in 60-mm

petri dishes, received2 mlof Hanksbalanced salt solution containing 5 ugof actinomycin D perml,

100 ,ug of cycloheximide per ml, and 5 JLCi of

[3H]uridine per ml. After 6 h at 37C, the

acid-precipitable radioactivity per culture was deter-mined as follows. The cell cultures were washed three times withHanks balanced salt solution and thensolubilizedwith 1% sodiumdodecylsulfatein 0.1MNaCland0.01MEDTA.Acid-insoluble

mate-rial wasprecipitatedwith 20%trichloroaceticacid,

and the precipitates were collected on glass-fiber filters(WhatmanGF/A)

Inactivation of VSV by UV light. Samples (2.5

ml)ofVSV inphosphate-bufferedsaline wereplaced

in60-mm glass petri dishes and irradiated with a

GeneralElectric 15-Wgermicidallampatadistance of50cm.The energyoutputatthisdistancewas12

ergs permm2asdeterminedbythemethodofJagger

(9).

RESULTS

Inhibition of PSR replication by VSV in

different cell lines. Because the inhibition of

thereplicationof PSRbyVSVhadbeen

demon-strated only in RK-13 cells (20), it was neces-sary todeterminewhether this inhibitory activ-ity of VSV was expressed in other cell lines. Various cell lines were infected with VSV and PSR at anMOI of 5 to 10; VSV wasaddedeither simultaneously with,or 2h after, PSR. After18

h at 37 C, total yields of PSR were determined in CE cells in the presence of VSV anti-body.

Table 1 shows that in all cell lines tested, VSV completely inhibited the replication of PSR. (Yields of PSR of104 PFU or lessper ml generallyrepresentedresidualvirusremaining after the washing procedure.) The ability of VSV to inhibitthe replication ofPSR was not

directly dependent on the yield of infectious VSV; this can be seenby comparing theyields of VSV in BHK-21 and RC-60 cells. In RC-60 cells, VSV producesan abortive infection that yields less than1PFU percell (15), whereasin

BHK-21 cells, VSV replicates to high titers. Evenunder theconditions ofanabortive infec-tion, VSV interfered with the replication of PSRwith the sameefficiency as inaproductive infection.

Dose response and estimation of the PSR-inhibiting activity of VSV. To estimate the PSR-inhibiting activity of VSV lysates, itwas necessarytodetermine thedose-response curve of the inhibition of PSR replication by VSV.

Serial twofold dilutions of the standard VSV preparationwereaddedtomonolayersof

BHK-21 cells simultaneously with PSR (MOI = 5).

Threecultureswereused for each dilution.

Fig-ure 1 shows the average yieldsof PSR plotted

againstthe number of PFU of VSVpercell. The

solid line is a theoretical

one-particle-per-cell

dose-responsecurvegeneratedfrom the Poisson distribution, whereas the closed circles

repre-sent experimentally determined values. As-sumingthatsolublefactorsare notresponsible

for the inhibitionof PSRreplication,thedatain

Fig. 1 are paradoxical. At an input MOI of

0.022, more than98% of thecells wouldnotbe infected, and yet theyield of PSRwasreduced byover 50%.These data suggest that thereare more viralparticles capable of inhibiting PSR replication than can be measured by plaque

formation. If the existence of these additional viral particles is given credence, the data are consistent with a mechanismwherebyoneVSV particleper cellissufficienttoprevent the

rep-licationof PSR.

Under the assumptionthatthe inhibition of PSRreplication byVSV is anall-or-none

phe-nomenon, onecanestimatethenumber of par-ticles inaVSVpoolcapable of inhibitingPSR

replication, since the inhibitionprocess follows

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TABLE 1. VSV-mediated interference with PSR replication in cells fromdifferentspecies Virus titer(18-hyield)

CellCell VSVTime of(h after PSR

superinfection

infection)with VSV(PFU/ml) P (PFU/ml) og decrease VSV(PFUml)PS~a(PFU/ml) (P8SR) MDCK(caninekidneyline)

CEM(primarychickenembryo

fibroblast)

RK-13 (rabbit kidney line)

L (mousefibroblast line)

BSC-1 (monkey kidney line)

BHK-21 (baby hamster kidney line)

VSVcontrol PSRcontrol

0 2

VSVcontrol

PSRcontrol

0 2

VSVcontrol PSRcontrol

0 2

VSVcontrol PSRcontrol

0 2

VSVcontrol PSRcontrol

0 2

VSVcontrol

PSRcontrol 0

2

1.6 x 108

1.2 x 108 7.7 x 107

3.4 x 108

4.0 x 104 1.6 x 105

1.6 x 108

4.4 x 107

1.4 x 108 6.4 x 104

1.0 x 108 3.5 x 105

2.3 x 106

4.4 x 108

2.6 x 106 3.2 x 104 6.5 x 106 3.0 x 103

8.2 x 107

8.6 x 107 1.4 x 108 1.3 x 108 1.6 x 108

2.8 x 108

1.2 x 106

3.0 x 103 5.0 x 103

9.4 x 107 4.0 x 103 3.0 x 103 2.6 x 108

1.6 x 108 2.6 x 108 2.0 x 103 2.8 x 108 7.0 x 103

RC-60(rabbit cornea line) VSVcontrol PSR control

0

1.8 x 105

1.2 x 109

7.1 x 103

a Yields of PSR of less than

104

represent virus remaining after thewashingprocedure.

aone-particle-per-cell doseresponse. The titer ofPSR-inhibitingparticles (PSRIP)was

deter-mined using thepredictions of the Poisson dis-tributionasdescribedbyMarcusandSekellick

(11). IfoneVSVparticlepercellissufficientto

preventPSRreplication,37% ofapopulationof

cells will yieldPSRatanMOI of VSV of1. A

virusinoculum that permitsaPSRyield of37%

ofcontrolvalues-would containasmanyPSRIP astherewerecells in theuninfected monolayer.

The titerofPSRIPcanbedeterminedby

know-ing the cell number and the dilution ofVSV

thatpermits37%ofcontrolyieldsofPSR. Us-ing this reasonUs-ing, the activities ofthe VSV preparation used toproducethe data inFig. 1

were calculatedtobe 1.4 x 1010PSRIP/ml and

3.1 x 108PFU/ml, aratioof 45 PSRIPto each PFU. The PSRIP/PFU ratio was also

deter-mined withVSVlysatesproducedinfour

differ-entcell lines.Althoughtheyieldof PFU varied by as much as100-fold, thePSRIP/PFU ratios wereidentical inall fourlysates, i.e., 32to64

PSRIP/PFU.

Effect of UV irradiation on the

PSR-in-hibiting activity of VSV. The ability ofVSV

inactivated by UV irradiation to inhibit PSR

replication wasdependent onthe cell line

em-ployed (Table 2). In RK-13 and BSC-1 cells, increasingirradiationcausedalossofabilityof

VSV to inhibit PSRreplication; irradiation of VSVfor 90sreduced the inhibition of PSRyield

byonly1 log. In contrast, in BHK-21 cells, the

same virus preparation still produced a 4-log

drop in PSRyield.Thereasonfor the different

efficiency ofUV-irradiated VSV in the various cell linesisnotclear. It ispossiblethat

process-ing ofUV-irradiatedVSV influences the inhibi-tionofPSR replicationto different degrees in

3.9 3.3

2.8 2.1

4.1

5.1

2.6 2.4

4.4

4.5

4.9 4.4

5.2 J. VIROL.

528 DUBOVI AND YOUNGNER

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0

z 0

U

0

I--z

10 .

U

a.

0.0

0.022 0.044 0.066 0.088

VSV(PFU PER CELL)

FIG. 1. Dose-response ofthe inhibition ofPSR

replication by infectiveVSV. Serial twofold dilutions ofVSVwereaddedsimultaneouslywithPSR (MOI

=5)tomonolayers ofBHK-21 cells.Infectedcultures

[image:4.503.43.237.56.281.2]

wereharvestedat18handassayed for infectivity of PSR in CE cells. Thesolid lineisatheoretical one-particle-per-celldose-responsecurvecalculated from the Poisson distribution, and the solidcircles repre-sentexperimentaldata.Controlyield ofPSR =3.0x 108PFUlml.

TABLE 2. InterferencewithPSRreplication: activity ofUV-irradiated VSVindifferentcelllines

Exposure 18-h PSR

Cell line time of VSV MOI(VSV)a yield(PFU/

toUV irra- Ml)

diation(s)

RK-13 6.0 x 108

0 20.0 1.8 x 105

30 0.0013 1.1 x 106 90 0.00013 4.1 x 107

BSC-1 2.1 x 10

0 26.0 4.3 x 104

30 0.0017 1.7 x 106

90 0.00017 1.8 x 107

BHK-21 6.4 x 108

0 17.0 2.8 x 104

30 0.0011 3.5 x 104 90 0.00011 4.1 x 104 aCalculated from the VSVinfectivity after expo-suretoUVirradiation.

RK-13 and BSC-1 cells, on one hand, and in BHK-21 cells, onthe other.

The abilityofUV-irradiated VSVto inhibit PSRreplicationinBHK-21cellswasexamined further using larger doses of UV light.

Expo-sureof

L,

VSVtoUV light for as longas300 s

stilldid notchangethe abilityofthe irradiated

virus to inhibit PSR replication in BHK-21 cells. With VSVgiven 300 sof irradiation, the

PSR yield in doublyinfected cells was 1.0 x 103 PFU/ml, in contrast to a control PSR yield of 6.0 x 107 PFU/ml. Since UV light primarily damages nucleicacids, these data suggest that

anactiveRNA genomeof VSV isnotneeded for the inhibition of PSR replication in BHK-21 cells and that the VSV virion may contain a

component capable of preventing the replica-tionof PSR.

Ifa virion component of VSV is capable of inhibiting the replication of PSR (3), a high MOI of VSV could mask the effect ofUV irra-diation. To eliminate this possibility,an experi-ment wasdesigned to assess the ability of lower multiplicities ofUV-irradiated VSV to inhibit the replicationof PSR. Serialtwofold dilutions ofunirradiated VSV (control) and VSV irradi-ated for 360s werecompared for their abilityto

inhibitPSR replication inBHK-21 cells. (The undiluted, unirradiated controlpreparation of VSVhadaninputMOI of 25.) Theability ofthe irradiated sample to inhibit PSR replication was rapidly lost upon dilution, whereas the unirradiated control still produced significant

inhibition at a 10-3dilution (Fig. 2). Approxi-mately800to1,000 timesmoreirradiatedvirus wasrequiredto achieve thesamelevel of

inhi-100

0

z

o UV-IRRADIATED

O 10 - (360SEC)

0

4-CONTROL

1.0

0 -I -2 -3 -4 -5

DILUTION OFVSV(LOG10)

FIG. 2. Effect of UV irradiation for 360 s on the ability of VSV to inhibit the replication of PSR in

BHK-21cells. Aliquots(2.5ml) of thestandardpool ofL,VSV diluted in phosphate-buffered saline were irradiated at a distance of 50 cm using a 15-w Gen-eral Electric germicidal lamp. Serial twofold dilu-tionsof unirradiated (control) and irradiated VSV

wereadded simultaneously with PSR (MOI=10) to monolayers of BHK-21 cells.Infected cultures were harvested at 18 h and assayed for infectivity of PSR

inCE cells. Control yield of PSR =1.6 x108 PFUI ml.

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530 DUBOVI AND YOUNGNER

bition produced by unirradiatedvirus. Whereas

oneunirradiated VSV particle percell was

suf-ficient to prevent the replication of PSR (Fig. 1), in the case of irradiated virus, a drastic

increase in the MOI was required to

compen-sate for the loss ofsynthetic activity that

re-sulted from irradiation.

Primary transcriptionby VSV and the inhi-bition of PSR replication. The effect of UV

irradiation on primary transcription by VSV wasdetermined using theinvivoassayof

Man-ders et al. (10). In striking agreement with Marcus and Sekellick (12), it was found that 264 ergs of UV irradiation were required to

reduce in vivo primary transcription to 37% of control values. The inactivation kinetics were

independentof the cell lineused; identical re-sults wereobtainedinBHK-21cells,acell line permissive for VSV replication, and in RC-60 cells, a cell line in which VSV undergoes an

abortive infection (15). As reported by Marcus and Sekellick (12), wefound that VSV infectiv-ity is five times more sensitive to UV irradia-tion than is primary transcription (slopes of -0.082 and -0.017, respectively; see Fig. 4).

Todetermine the UV inactivation rateof the PSR-inhibiting activity of VSV in BHK-21 cells, the standard pool of

L,

VSV was diluted

in phosphate-buffered saline to give an input MOI of 1.0. This low MOI was necessary be-cause ofthe ability of UV-irradiated VSV, at

high MOI toinhibit PSR replicationin BHK-21

cells (Table 2). Similar experiments were done usingRC-60 and RK-13 cells; however, higher MOIs (19 and 45, respectively) were used be-causethese cells are less sensitive than BHK-21 cells to inactivated VSV (Table 2). Virus was

irradiated as previously described and tested for itsabilitytointerfere with thereplication of PSR. The results (Fig. 3) show that the inacti-vation rate of the PSR-inhibiting activity of

L,

VSV is not significantly different in the three cell lines tested. (Loss of interference is shown

by an increase inyields of PSR indoubly

in-fected cells.) No direct comparisons can be made between the doses of UV light and the levels ofinhibition of PSR replication in these cell linessincethe MOIare different. Figure 4 compares the inactivation of the following ac-tivities ofVSV: infectivity, primary transcrip-tion, and inhibition of PSR replication in

BHK-21 cells. The loss ofinhibition of PSR replica-tionby VSV was moresensitive than primary transcription to low doses of UV irradiation. However, VSV irradiated for 110 s still in-hibited PSR replication by >90% in BHK-21 cells.

Inhibition of PSR replication by ts mutants

ofVSVat 39.5C.Atleast onerepresentative of

J. VIROL.

each of the five complementation groups of VSVIND identified by Flamand and Pringle (5) wastested for its ability to inhibit the replica-tionof PSR at the nonpermissive temperature (39.5C). The RNA phenotypes at 39.5 C of these mutants agreed with published reports, and the leak and revertant yields of the

mu-tants wereless than 0.002 PFU per cell. Monolayers of BHK-21 cells grown in welled trays (16-mm wells) were infected simultane-ously with PSR (MOI = 10) and VSV (MOI =

5).After1h at4C, the inoculum wasremoved, and the monolayers were washed three times with Eagle minimal essential medium. The welled trays were sealed in plastic bags and immersed in water baths at 39.5 C. Infected fluidsand cells were havested at18 hand as-sayedaspreviously described.

Withone exception, complementation group I mutants, which show little or no primary transcription at 39.5 C, did not inhibit the rep-lication of PSR at this temperature (Table3). Mutant tsO-5 behaveddifferently than the other group I mutants. Interestingly, Flamand and

Bishop(4)reportedthattsO-5producedprimary

9

8

E/

6.. 7

U-0.

0

-J

4

6

0 20 40 60 80 100 120 140

UV-IRRADIATION (SEC)

FIG. 3. Kinetics of inactivation of the

PSR-in-hibitingactivityofVSVindifferent cell lines. Sam-plesof L, VSV, UV irradiated as described in the

legendtoFig. 2,weretestedfor theirabilitytoinhibit the replication of PSR inRC-60 cells (A),BHK-21 cells(0), and RK-13 cells (0). MOI of VSV: BHK-21 cells=1; RK-13cells=45;RC-60 cells=19.Control yieldsof PSR(PFUlml): RC-60 =3.0 x108; RK-13

=1.9 x108; BHK-21 =4.6 x108.

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- 6

0

E

5

tLA

4

3

0 20 40 60 80

UVIRRADIATION (SEC)

FIG. 4. Comparison ofthe inactiva

radiation of infectivity, primary tran PSR-inhibiting activity of VSV. Aliqz

were irradiated and tested for infect

mary transcription (0), and the inh replication(@).Primary transcription asfollows. Monolayers ofBHK-21 cell atanMOIof25(calculated fromtheib unirradiated sample). After adsorpt 4C,eachmonolayerin60-mmpetridi ml ofHanks balancedsaltsolutioncon actinomycinDperml,100 pgofcyc

ml, and 5 pCi of [3H]uridineper mi tureswereharvestedafter6 hat39C

forradioactivity determinations.Thei curve (BHK-21 cells) is takenfrom I

yield ofPSRin BHK-21cells=4.6x1

100 PSR replication. The RNA+ mutants tsO-23

(groupIll)and ts0-45 (group V)interferedwith PSR replicationtothesameextent.Additional representativesof each complementationgroup

will have to be examined before any

correla-°o tionscanbemade betweenthe knowntsdefects

_ and the ability to inhibit PSR replication

be-CO cause of the possible variations in phenotypic

expression within the same complementation group(12).

DISCUSSION

Thedetection ofanactivityofavirus

prepa-ration that is quantitatively greater than the concentration of PFU always raises the

ques-tion of thenatureofthe virusparticle

responsi-2 ble for theactivity.Dothese virusparticlesfail

, to produce plaques because ofa defect in the

= virion or arethey victims of chance? At pres-A ent, thisquestionhasnot beendefinitively

an-t swered, partly because eachvirusstudied

prob-ably presents a different mosaic of these two

possibilities. ForVSV, the physical particle-to-PFU ratio has been reported as 40 to 1 (6) or

between 73 to1and194to1(13).Itis not known

100 120 whether the particles that fail to produce

plaquesaredefective.

tionby UV ir- In theirstudy of cellkilling by VSVparticles, iscription, and Marcusand Sekellick (11) detected cellkilling uotsof L, VSV particles thatwere usually ingreater

concen-tivity (A), pri- tration thanPFU. Although thetwoactivities ibition of PSR could not be separated in sucrose gradients,

was measured they assumed that cell-killingparticles in

ex-gswereinfected cessof PFUweredefectiveandreferredtothem

nfectivityofthe as"defectivecell-killing particles."

ishes

received2 The present report demonstrates that the

tamningr 5 g of number ofVSVparticles capable ofinhibiting

loheximideper the replication of PSR (PSRIP) exceeds the l.Infectedcul- number of PFU by a factor of 32 to 64. This

andprocessed value is in close agreement with the

particle-to-PSR inhibition PFU ratios reported previously (6, 13). The

Fig.3. Control PSRIP were not designated defective because

rurr u/ml.

transcripts from the inputgenomewithan

effi-ciency equal to wild-typevirus at39.5C.

Per-hapsthis level of RNAsynthesisissufficientto

produce inhibition of the replication of PSR.

Surprisingly, therewas no difference inthe

inhibition of PSRreplication bytheRNA-

mu-tanttsO-52 (groupII)and the RNA+mutant

tsO-23 (group III). Mutant tsO-23, whichcancarry

out primary transcription and replication at

39.5C, synthesized 60 times more RNA than

tsO-52, which showsonly primary transcription

at the nonpermissive temperature

(unpub-lished observation). Mutant tsG-41, another

RNA-mutantthatisdefectiveinRNA replica-tionat39.5C,hadareducedcapacityto inhibit

TABLE 3. Ability oftsmutantsof VSVtoinhibit the replication of PSRinBHK-21 cells at 39.5C VSV MU- Complemen- RNA syn- PSyil

V~tanta station thesisat39.5

(PSFU/iel)

PSR control 1.5 x 108

tsO-5 I - 6.1 x 104

tsG-11 I - 1.3 x 108

tsG-13 I - 1.3 x 108

tsG-16 I - 1.1 x 108

tsO-52 II - 4.3 x 105

tsO-23 III + 1.8 x 105

tsG-41 IV - 3.8 x 107

tsO-45 V + 2.2 x 105

L, VSV Wildtype + 2.4 x 104

aMOI of5.

8,^

Cl

M

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532 DUBOVI AND YOUNGNER

there isnodirect evidence that this is thecase.

The high ratio of PSRIPtoPFU isnotsufficient

justificationforconcluding that the PSRIPare

defective. Since therearemultiplefactors that caninfluenceaPFUassay,theconcentration of

PFU detected under aparticular set of

condi-tions must be considered a relative titer. For

example,VSV undergoesanabortiveinfection

inRC-60 cells (15), andaPFUassayof VSV in

these cells would be negative. However, a

PSRIPassayinRC-60cells would revealahigh

concentration ofVSV particles. Werenoother

cell lines available, one might be inclined to designateallthe PSRIP defective. The availa-bility of cell linesmorepermissive than the

RC-60line makes this assumption untenable. Per-haps when more permissive cell lines for VSV are found, the PSRIP-to-PFU ratio will

ap-proach 1.

At present, we have no direct evidence to

suggest that PSRIP and defective cell-killing

particles are the same particles. However, it

appearsunlikely thateveryinhibitoryactivity of VSVis dueto aunique virus particle. Both

PSRIP and defective cell-killing particle

activ-ities showaone-particle-per-celldoseresponse,

suggesting thatsynthesis ofaviralproduct is necessary for inhibition. Also, atthe

nonper-missive temperature, complementationgroupI

mutants atlow MOIareunabletoexpresscell

killing(12)orinhibitionof PSR replication

(Ta-ble 3). In addition, group I mutants at high MOI cankillcells (12) and inhibit the

replica-tion of PSR(unpublishedobservation).The UV

inactivation kineticsof cellkilling (12)and

in-hibitionof PSRreplicationby VSV (Fig. 3)are

different,but this probably reflects differences

in the methods of quantitating the inhibitory

activities. Cells are scored as either dead or

alive, but the inhibition of PSR replicationcan rangefroma5-logdecrease in virus yieldtono

decrease. The complex UV inactivationcurveof

PSR inhibition by VSV may reflect different

levels of inhibition thatdependontheextentof

the VSV replication cycle completed by the ir-radiated particle. For complete inhibition of PSR replication by VSV at low MOI, a fully

infectiousparticle is required foramplification

oftheinputgenometooccur.This amplification process exponentially increases the amount of viralproducts in the cell and results inarapid

increase inthe putative inhibitory component

thatrapidlypreventsPSRreplication. Without amplification of the input genome, primary

transcriptioncanincrease viral proteinsonly in alinearmanner, resulting inaslowerbuildup

oftheinhibitorycomponents.Inthisway,

syn-thesis of some PSR virions could occur. The

need foramplificationtorapidlyincrease VSV

J. VIROL.

products within the doubly infected cell could explainthe high sensitivity ofPSR inhibition

byVSVtosmall doses of UVirradiation. The data concerning the PSR-inhibiting

ac-tivity of UV-irradiated VSV in BHK-21 cells stronglysupportstheidea ofaninhibitory com-ponentin the virion ofVSV, as has been

pro-posed innumerous reports. Inastudyof

cyto-toxicity by UV-irradiated VSV, Cantell et al. (2)showed thatUV-irradiatedVSV killed cells inthe absence ofdetectableviral-directed

pro-teinsynthesis.Cellkilling requiredahigh MOI

of irradiated VSV, and the cytotoxic factor could not beseparatedfrom the virusparticle.

HuangandWagner (8) demonstratedthat

UV-irradiated VSV inhibited host RNA synthesis inKrebs-2mouseascitescells,whereas Yaoiet

al. (19) reporteda similarphenomenon in CE cells.MarcusandSekellick (12) suggestedthat residualprimary transcription byVSVmaybe

responsible fortheinhibitionof host RNA syn-thesisby UV-irradiatedVSV.However,Huang and Wagner (8)employed dosesofUV irradia-tionthat wouldhave completely abolishedeven

primary transcription, and their data strongly supporttheidea of aninhibitory component in

the virion of VSV. Additional support forthis

conceptcomesfrom thereportsof(i)Wertz and

Youngner (17)ontheinhibition ofhostprotein synthesisby UV-irradiated VSV, (ii) Yamazaki

and Wagner (18)onthecytotoxicity ofVSV in

interferon-treated cells, and (iii) Huang etal.

(7)andDubovi andYoungner (3)onthe

inhibi-tion of host RNAsynthesis by unirradiatedand

UV-irradiated DI particles ofVSV.

An inhibitory component in the virion of

VSVmayalso beresponsible for the inhibition

ofthe replication of PSR by inactivatedVSV. Aswith cell killing and the inhibition ofhost RNA and protein synthesis, inhibition ofPSR

replication by UV-irradiated VSV requires a

high MOI ofVSV.The datainFig. 2showthat

approximately 800 to 1,000 times more inacti-vated particles than infectious particles are

necessarytoproduce equivalentlevels of inhi-bition. In addition, at thenonpermissive

tem-perature, complementation group I mutants, whichareunabletoinhibit PSR replicationat a

low MOIof5 (Table3),doinhibit PSR

replica-tion at ahighMOI of 60 (unpublished

observa-tion).The inhibitionofPSRreplicationby

inac-tivated VSV and infectious VSV is quantita-tively different because of the ability of the infectious particletoamplify theinput genome.

Flamand and Bishop(4)estimated thatasingle

cell can yield 104 to 105 viral particles. If a proteincomponentof VSV isresponsible for the inhibition ofPSRreplication, itmay be neces-sary toreach athresholdconcentration ofthis

on November 10, 2019 by guest

http://jvi.asm.org/

(8)

protein before interference is expressed. Since heavily irradiated B particles possess little, if

any, synthetic activity, it seemslikely that a

high MOIofirradiatedviruswould berequired to introduce sufficient viral protein to cause inhibition of PSRreplication. This situation is somewhat analogous to cell fusion by para-myxoviruses. For fusion-from-without, a high multiplicity of inactivated virus is necessary, but forfusion-from-within, oneinfectious parti-cle per cell issufficient. (1).

Data presented in an accompanying report (3)concerningtheability of DIparticlesof VSV toinhibitPSR replication, host RNAsynthesis, and host proteinsynthesisstrongly support the

concept of an inhibitory component in the

vir-ion of VSV. These data, along with published reports, have led us to propose that a virion component of VSV may be responsible for all

the inhibitory activities ofVSV, except homolo-gousinterference.

ACKNOWLEDGMENTS

Thisinvestigation was supported byPublic Health Ser-viceresearch grant AI-06264 from the National Institute of AllergyandInfectious Diseases.

LITERATURE CITED

1. Bratt, M. A., and W. R. Gallaher. 1972. Biological parameters of fusion from within and fusion from without, p. 383-406.In C. F. Fox(ed.), Membrane research. Academic PressInc.,New York.

2. Cantell, K., Z. Skurska, K. Paucker, andW. Henle. 1962. Quantitative studies on viral interference in suspendedLcells. II. Factors affectinginterference by UV-irradiatedNewcastledisease virus against

ve-sicular stomatitis virus.Virology17:312-323. 3. Dubovi, E. J.,andJ. S. Youngner. 1976. Inhibition of

pseudorabiesvirusreplication byvesicular stomatitis virus. II. Activity ofdefective interfering particles. J. Virol. 18:534-541.

4. Plamand, A., and D. H. L.Bishop. 1973. Primaryin

vivotranscription ofvesicular stomatitis virus and temperature-sensitive mutants of fivevesicular sto-matitis virus complementation groups. J. Virol. 12:1238-1252.

5. Flamand, A., and C.R.Pringle.1971.Thehomologies of spontaneous and induced temperature-sensitive mutantsofvesicularstomatitis virusisolatedinchick

embryo andBHK-21cells. J. Gen. Virol. 11:81-85. 6. Howatson, A.F., and G. F. Whitmore. 1962. The

devel-opmentand structureof vesicular stomatitis virus. Virology 16:466-478.

7. Huang, A. S., J. W. Greenawalt, and R. R. Wagner. 1966. Defective Tparticles of vesicular stomatitis vi-rus. I. Preparation, morphology, and some biologic properties. Virology 30:161-172.

8. Huang, A.S., and R. R. Wagner. 1965. Inhibition of cellularRNA synthesis by nonreplicating vesicular stomatitis virus. Proc. Natl. Acad. Sci. U.S.A. 54:1579-1584.

9. Jagger, J. 1961. A small and inexpensive ultraviolet dose-rate meter useful in biological experiments. Ra-diat. Res. 14:394-403.

10. Manders, E. K.,J. G. Tilles, and A. S. Huang. 1972. Interferon-mediated inhibition of virion-directed transcription. Virology 49:573-581.

11. Marcus, P. I., and M.J. Sekellick. 1974. Cell killing by viruses.I. Comparison ofcell-killing, plaque forma-tion and defectiveinterfering particles of vesicular stomatitis virus. Virology 57:321-338.

12. Marcus, P.I.,and M. J. Sekellick. 1975. Cell killingby viruses.II.Cell killing by vesicular stomatitis virus: arequirement forvirion-derived transcription. Virol-ogy63;176-190.

13. McCombs, R. M., M. Benyesh-Melnick, and J. P. Brunschwig. 1966. Biophysical studies of vesicular stomatitis virus. J. Bacteriol. 91:803-812.

14. McSharry, J. J., and R. Benzinger. 1970. Concentration andpurification of vesicular stomatitis virus by poly-ethylene glycol precipitation. Virology 40:745-746. 15. Thacore, H. R., andJ. S. Youngner. 1975. Abortive

infection of a rabbit cornea cell line by vesicular stomatitis virus: conversion toproductiveinfectionby superinfectionwith vaccinia virus. J. Virol. 16:322-329.

16. Wertz, G. W., and J. S. Youngner. 1970. Interferon production and inhibition of hostsynthesis incells infected with vesicular stomatitis virus. J. Virol. 6:476-484.

17. Wertz,G. W.,and J. S. Youngner. 1972. Inhibition of proteinsynthesis in L cells infected with vesicular stomatitisvirus. J.Virol.9:85-89.

18. Yamazaki, S., and R. R. Wagner. 1970. Action of inter-feron: kineticsand differential effects on viral func-tions.J. Virol. 6:421-429.

19. Yaoi, Y., H. Mitsui, and M. Amano.1970.Effect of UV-irradiated vesicular stomatitis virus on nuceleicacid synthesisinchickembryocells. J. Gen. Virol. 8:165-172.

20. Youngner, J. S., H. Thacore, and M.Kelly.1972. Sensi-tivity ofribonucleic acid and deoxyribonucleic acid viruses todifferentspecies ofinterferonincell cul-tures. J.Virol. 10:171-178.

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Figure

TABLE 1. VSV-mediated interference with PSR replication in cells from different species yield)
FIG.1.particle-per-cell108PSRsentofthereplicationwere= 5) VSV Dose-response of the inhibition of PSR by infective VSV
FIG. 3.yieldsplescellscellslegendhibitingthe= 1.9 Kinetics of inactivation of the PSR-in- activity of VSV in different cell lines
FIG. 4.radiation Comparison of the inactiva of infectivity, primary traniscription,

References

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