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
virionproducts 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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[image:3.503.61.455.74.438.2]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 sstilldid 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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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 dilutedin 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 shownby 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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[image:5.503.279.468.337.571.2]- 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 thetamningr 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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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
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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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