JOURNAL OF VIROLOGY, May 1976, p. 526-533 Copyright © 1976 American Society for Microbiology
Vol. 18, No. 2 Printed in U.SA.
Inhibition of Pseudorabies Virus Replication by Vesicular Stomatitis Virus I. Activity of Infectious and Inactivated B Particles EDWARD J. DUBOVI' AND JULIUS S. YOUNGNER* Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received for publication 5 December 1975
Infectious B particles of vesicular stomatitis virus (VSV) are capable of inhibiting the replication of pseudorabies virus (PSR) in a variety of cell lines. Even under conditions of an abortive infection in a continuous line of rabbit cornea cells (RC-60), B particles interfere with the replication of PSR with high efficiency. Particle per cell dose-response analysis of B particle populations revealed that the number of VSV particles capable of inhibiting PSR replication exceeds the number of PFU by a factor of 32 to 64. When B particles are treated with UV irradiation, a drastic increase in the multiplicity of infection is required to inhibit PSR replication. Whereas one infective B particle per cell is sufficient to prevent replication of PSR, 800 to 1,000 VSV particles rendered noninfective by UV irftdiation are required to compensate for the loss of VSV synthetic activity that results from irradiation. Temperature-sensitive mutants representing five complementation groups of VSV were tested at low multiplicities of infection for their effect on PSR replication at the nonpermissive temperature. Generally, the ability of the different complementation groups to amplify virion products at the nonpermissive temperature is associated with their ability to inhibit PSR replication. These results imply that at low multiplicities of infection, amplification of infecting VSV components is necessary for inhibition of PSR replication, but at high multiplicities of infection with VSV, a virion component can prevent PSR replication in the absence of de novo VSV RNA or protein synthesis. Youngner et al. (20) reported that vesicular stomatitis virus (VSV) completely inhibited the replication of pseudorabies virus (PSR) in a continuous line of rabbit kidney cells (RK-13). Rabbit cells in culture are unusual in that pretreatment of such cells with interferon fails to inhibit the replication of DNA viruses, whereas inhibition of RNA viruses is normal (20). Therefore, in RK-13 cells pretreated with interferon, VSV was unable to suppress the replication of PSR. This suggested that the inhibition of PSR replication of VSV is caused by a product synthesized during the VSV replication cycle. The present study reports the conditions necessary for inhibition of PSR replication by VSV in a variety of cell lines. VSV rendered noninfective by UV irradiation and temperature-sensitive (ts) mutants representing five complementation groups of VSV were tested for their ability to inhibit the replication ofPSR.
MATERIALS AND METHODS
Cells. Primary chicken embryo (CE) cells, mouse L cells (clone 929), a line (BHK-21) of hamster kidney cells, and a line (MDCK) of canine kidney cells were propagated in Eagle minimal essential medium plus 4% calf serum. Cell lines of rabbit kidney cells (RK-13), rabbit cornea cells (RC-60), and monkey kidney cells (BSC-1) were grown in MEM plus 10% fetal calf serum. Viruses. The large-plaque mutant of VSVIND (L, VSV) described by Wertz and Youngner (16) was grown in BHK-21 cells. Monolayers in 32-ounce (ca. 960-ml) culture bottles were infected with less than 0.01 PFU per cell to avoid the production of defective interfering (DI) particles. Analysis of [3H]uridinelabeled viral particles by sucrose gradients failed to detect DI particles in lysates produced under these conditions. A single pool of L, VSV was used throughout this study. Virus for this pool was concentrated by polyethylene glycol 6000 as described by McSharry and Benzinger (14) and was partially purified by pelleting the virus through a 50% glycerol cushion. Virus prepared in this manner was stored at -70 C. Temperature-sensitive mutants of VSVIND were obtained from R. R. Wagner. Well-
1 Present address: Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, Va.
22901.
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INHIBITION OF PSR BY VSV B PARTICLES. I.
isolated plaques from terminal dilutions incubated at 32 C were used to produce pools of each mutant in BHK-21 cells at 32 C. The efficiency of plating (39.5/ 32) in CE cells was less than 10-4 for all clones used in this study. PSR originally 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. Cycloheximide was purchased from Upjohn Co., Kalamazoo, Mich., and actinomycin D was obtained through the courtesy of H. B. Woodruff of Merck, Sharpe and Dohme. Double infections with VSV and PSR. Unless otherwise stated, VSV and PSR were added simultaneously to cell cultures in 60-mm petri dishes at an input multiplicity of infection (MOT) of 5 to 10 for each virus. After an adsorption period of 2 h at 37 C in a volume of 1.0 ml, the inoculum was removed by three washes with Eagle minimal essential medium. Three milliliters of growth medium was added, and the cultures were incubated for 18 to 20 h at 37 C. Fluid plus cells were harvested, and the suspension was sonicated to release cell-associated virus. To titrate the PSR, it was necessary to neutralize the infectivity of VSV. This was accomplished by adding anti-VSV antibody to the 10-2 dilution of the virus sample and incubating at 37 C for 1 h. The few VSV plaques that occasionally survived this treatment could easily be discriminated 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 polymerase of VSV was done according to Manders et al. (10). UV-irradiated or unirradiated VSV was added to monolayers of BHK-21 or RC-60 cells at an MOI of 25 (calculated from the unirradiated sample) for 1 h at 4 C. After adsorption, each monolayer, in 60-mm petri dishes, received 2 ml of Hanks balanced salt solution containing 5 ug of actinomycin D per ml, 100 ,ug of cycloheximide per ml, and 5 JLCi of [3H]uridine per ml. After 6 h at 37 C, the acidprecipitable radioactivity per culture was determined as follows. The cell cultures were washed three times with Hanks balanced salt solution and then solubilized with 1% sodium dodecyl sulfate in 0.1 M NaCl and 0.01 M EDTA. Acid-insoluble material was precipitated with 20% trichloroacetic acid, and the precipitates were collected on glass-fiber filters (Whatman GF/A) Inactivation of VSV by UV light. Samples (2.5 ml) of VSV in phosphate-buffered saline were placed in 60-mm glass petri dishes and irradiated with a General Electric 15-W germicidal lamp at a distance of 50 cm. The energy output at this distance was 12 ergs per mm2 as determined by the method of Jagger (9).
RESULTS Inhibition of PSR replication by VSV in different cell lines. Because the inhibition of the replication of PSR by VSV had been demon-
527
strated only in RK-13 cells (20), it was necessary to determine whether this inhibitory activity of VSV was expressed in other cell lines. Various cell lines were infected with VSV and PSR at an MOI of 5 to 10; VSV was added either simultaneously with, or 2 h after, PSR. After 18 h at 37 C, total yields of PSR were determined in CE cells in the presence of anti-VSV antibody. Table 1 shows that in all cell lines tested, VSV completely inhibited the replication of PSR. (Yields of PSR of 104 PFU or less per ml generally represented residual virus remaining after the washing procedure.) The ability of VSV to inhibit the replication of PSR was not directly dependent on the yield of infectious VSV; this can be seen by comparing the yields of VSV in BHK-21 and RC-60 cells. In RC-60 cells, VSV produces an abortive infection that yields less than 1 PFU per cell (15), whereas in BHK-21 cells, VSV replicates to high titers. Even under the conditions of an abortive infection, VSV interfered with the replication of PSR with the same efficiency as in a productive infection. Dose response and estimation of the PSRinhibiting activity of VSV. To estimate the PSR-inhibiting activity of VSV lysates, it was necessary to determine the dose-response curve of the inhibition of PSR replication by VSV. Serial twofold dilutions of the standard VSV preparation were added to monolayers of BHK21 cells simultaneously with PSR (MOI = 5). Three cultures were used for each dilution. Figure 1 shows the average yields of PSR plotted against the number of PFU ofVSV per cell. The solid line is a theoretical one-particle-per-cell dose-response curve generated from the Poisson distribution, whereas the closed circles represent experimentally determined values. Assuming that soluble factors are not responsible for the inhibition of PSR replication, the data in Fig. 1 are paradoxical. At an input MOI of 0.022, more than 98% of the cells would not be infected, and yet the yield of PSR was reduced by over 50%. These data suggest that there are more viral particles 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 mechanism whereby one VSV particle per cell is sufficient to prevent the replication of PSR. Under the assumption that the inhibition of PSR replication by VSV is an all-or-none phenomenon, one can estimate the number of particles in a VSV pool capable of inhibiting PSR replication, since the inhibition process follows
528
J. VIROL.
DUBOVI AND YOUNGNER
TABLE 1. VSV-mediated interference with PSR replication in cells from different species Virus titer (18-h yield)
with VSV (PFU/ml) P Time (h of after superinfection (PFU/ml) PSR infection) VSV VSV (PFUml) PS~a(PFU/ml)
Cell Cell
1.6 x 108
VSV control PSR control
MDCK (canine kidney line)
1.2 x 108 7.7 x 107
0
2
CEM (primary chicken embryo fibroblast)
RK-13 (rabbit kidney line)
L (mouse fibroblast line)
BSC-1 (monkey kidney line)
BHK-21 (baby hamster kidney line)
RC-60 (rabbit cornea line) a
Yields of PSR of less than
VSV control
1.6 x 108
PSR control 0 2
1.4 x 108 1.0 x 108
VSV control PSR control 0 2
2.3 x 106
VSV control PSR control 0 2
8.2 x 107
VSV control PSR control 0 2
1.3 x 108
VSV control
2.6 x 108
PSR control 0 2
2.6 x 108 2.8 x 108
8.6 x 107 1.4 x 108
1.6 x 108 2.8 x 108
(P8SR)
3.4 x 108 4.0 x 104 1.6 x 105
3.9 3.3
4.4 x 107 6.4 x 104 3.5 x 105
2.8 2.1
4.4 x 108 3.2 x 104 3.0 x 103
4.1 5.1
1.2 x 106 3.0 x 103 5.0 x 103
2.6 2.4
9.4 x 107 4.0 x 103 3.0 x 103
4.4 4.5
1.6 x 108 2.0 x 103 7.0 x 103
4.9 4.4
1.2 x 109 7.1 x 103
5.2
1.8 x 105
VSV control PSR control 0
104 represent virus remaining after the washing procedure.
one-particle-per-cell dose response. The titer of PSR-inhibiting particles (PSRIP) was determined using the predictions of the Poisson distribution as described by Marcus and Sekellick (11). If one VSV particle per cell is sufficient to prevent PSR replication, 37% of a population of cells will yield PSR at an MOI of VSV of 1. A virus inoculum that permits a PSR yield of 37% of control values-would contain as many PSRIP as there were cells in the uninfected monolayer. The titer of PSRIP can be determined by knowing the cell number and the dilution of VSV that permits 37% of control yields of PSR. Using this reasoning, the activities of the VSV preparation used to produce the data in Fig. 1 were calculated to be 1.4 x 1010 PSRIP/ml and 3.1 x 108 PFU/ml, a ratio of 45 PSRIP to each PFU. The PSRIP/PFU ratio was also determined with VSV lysates produced in four differa
2.6 x 106 6.5 x 106
og decrease
ent cell lines. Although the yield of PFU varied as much as 100-fold, the PSRIP/PFU ratios were identical in all four lysates, i.e., 32 to 64
by
PSRIP/PFU. Effect of UV irradiation on the PSR-inhibiting activity of VSV. The ability of VSV inactivated by UV irradiation to inhibit PSR replication was dependent on the cell line employed (Table 2). In RK-13 and BSC-1 cells, increasing irradiation caused a loss of ability of VSV to inhibit PSR replication; irradiation of VSV for 90 s reduced the inhibition of PSR yield by only 1 log. In contrast, in BHK-21 cells, the same virus preparation still produced a 4-log drop in PSR yield. The reason for the different efficiency of UV-irradiated VSV in the various cell lines is not clear. It is possible that processing of UV-irradiated VSV influences the inhibition of PSR replication to different degrees in
529 sure of L, VSV to UV light for as long as 300 s still did not change the ability of the irradiated virus to inhibit PSR replication in BHK-21 cells. With VSV given 300 s of irradiation, the PSR yield in doubly infected cells was 1.0 x 103
INHIBITION OF PSR BY VSV B PARTICLES. I.
VOL. 18, 1976
0
z
0 U
0
I-z
10 .
U
a.
0.0
0.022
0.044 0.066 VSV (PFU PER CELL)
0.088
FIG. 1. Dose-response of the inhibition of PSR replication by infective VSV. Serial twofold dilutions of VSV were added simultaneously with PSR (MOI = 5) to monolayers of BHK-21 cells. Infected cultures were harvested at 18 h and assayed for infectivity of PSR in CE cells. The solid line is a theoretical oneparticle-per-cell dose-response curve calculated from the Poisson distribution, and the solid circles represent experimental data. Control yield ofPSR = 3.0 x 108 PFUlml. TABLE 2. Interference with PSR replication: activity of UV-irradiated VSV in different cell lines Exposure 18-h PSR time of VSV (PFU/ Cell line to UV irra- MOI (VSV)a yieldMl) diation (s)
RK-13 0 30 90
20.0 0.0013
0 30 90
26.0 0.0017 0.00017
0.00013
BSC-1
6.0 x 108 1.8 x 105 1.1 x 106
4.1 x 107
2.1 4.3 1.7 1.8
PFU/ml, in contrast to a control PSR yield of 6.0 x 107 PFU/ml. Since UV light primarily damages nucleic acids, these data suggest that an active RNA genome of VSV is not needed for the inhibition of PSR replication in BHK-21 cells and that the VSV virion may contain a component capable of preventing the replication of PSR. If a virion component of VSV is capable of inhibiting the replication of PSR (3), a high MOI of VSV could mask the effect of UV irradiation. To eliminate this possibility, an experiment was designed to assess the ability oflower multiplicities of UV-irradiated VSV to inhibit the replication of PSR. Serial twofold dilutions of unirradiated VSV (control) and VSV irradiated for 360 s were compared for their ability to inhibit PSR replication in BHK-21 cells. (The undiluted, unirradiated control preparation of VSV had an input MOI of 25.) The ability of the irradiated sample to inhibit PSR replication was rapidly lost upon dilution, whereas the unirradiated control still produced significant inhibition at a 10-3 dilution (Fig. 2). Approximately 800 to 1,000 times more irradiated virus was required to achieve the same level of inhi100 0
z
o O 0
UV-IRRADIATED (360 SEC)
10 -
4-CONTROL 1.0
x 10
x 104 x 106 x 107
6.4 x 108 2.8 x 104 17.0 0 3.5 x 104 0.0011 30 4.1 x 104 90 0.00011 a Calculated from the VSV infectivity after exposure to UV irradiation.
BHK-21
RK-13 and BSC-1 cells, on one hand, and in BHK-21 cells, on the other. The ability of UV-irradiated VSV to inhibit PSR replication in BHK-21 cells was examined further using larger doses of UV light. Expo-
0
-I
-2
-3
DILUTION OF VSV
-4
-5
(LOG10)
FIG. 2. Effect of UV irradiation for 360 s on the ability of VSV to inhibit the replication of PSR in BHK-21 cells. Aliquots (2.5 ml) of the standard pool of L, VSV diluted in phosphate-buffered saline were irradiated at a distance of 50 cm using a 15-w General Electric germicidal lamp. Serial twofold dilutions of unirradiated (control) and irradiated VSV were added simultaneously with PSR (MOI = 10) to monolayers of BHK-21 cells. Infected cultures were harvested at 18 h and assayed for infectivity of PSR in CE cells. Control yield of PSR = 1.6 x 108 PFUI ml.
530
DUBOVI AND YOUNGNER
bition produced by unirradiated virus. Whereas one unirradiated VSV particle per cell was sufficient to prevent the replication of PSR (Fig. 1), in the case of irradiated virus, a drastic increase in the MOI was required to compensate for the loss of synthetic activity that resulted from irradiation. Primary transcription by VSV and the inhibition of PSR replication. The effect of UV irradiation on primary transcription by VSV was determined using the in vivo assay of Manders 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 independent of the cell line used; identical results were obtained in BHK-21 cells, a cell 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), we found that VSV infectivity is five times more sensitive to UV irradiation than is primary transcription (slopes of -0.082 and -0.017, respectively; see Fig. 4). To determine the UV inactivation rate of 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 because of the ability of UV-irradiated VSV, at high MOI to inhibit PSR replication in BHK-21 cells (Table 2). Similar experiments were done using RC-60 and RK-13 cells; however, higher MOIs (19 and 45, respectively) were used because these cells are less sensitive than BHK-21 cells to inactivated VSV (Table 2). Virus was irradiated as previously described and tested for its ability to interfere with the replication of PSR. The results (Fig. 3) show that the inactivation 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 in yields of PSR in doubly infected cells.) No direct comparisons can be made between the doses of UV light and the levels of inhibition of PSR replication in these cell lines since the MOI are different. Figure 4 compares the inactivation of the following activities of VSV: infectivity, primary transcription, and inhibition of PSR replication in BHK21 cells. The loss of inhibition of PSR replication by VSV was more sensitive than primary transcription to low doses of UV irradiation. However, VSV irradiated for 110 s still inhibited PSR replication by >90% in BHK-21 cells. Inhibition of PSR replication by ts mutants of VSV at 39.5 C. At least one representative of
J. VIROL.
each of the five complementation groups of VSVIND identified by Flamand and Pringle (5) was tested for its ability to inhibit the replication of PSR at the nonpermissive temperature (39.5 C). The RNA phenotypes at 39.5 C of these mutants agreed with published reports, and the leak and revertant yields of the mutants were less than 0.002 PFU per cell. Monolayers of BHK-21 cells grown in welled trays (16-mm wells) were infected simultaneously with PSR (MOI = 10) and VSV (MOI = 5). After 1 h at 4 C, the inoculum was removed, 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 fluids and cells were havested at 18 h and assayed as previously described. With one exception, complementation group I mutants, which show little or no primary transcription at 39.5 C, did not inhibit the replication of PSR at this temperature (Table 3). Mutant tsO-5 behaved differently than the other group I mutants. Interestingly, Flamand and Bishop (4) reported that tsO-5 produced primary 9
8
E/ 6..
7
U-
0.
0 -J
4
6
0
20
40 60 80 100 UV- IRRADIATION (SEC)
120
140
FIG. 3. Kinetics of inactivation of the PSR-inhibiting activity of VSV in different cell lines. Samples of L, VSV, UV irradiated as described in the legend to Fig. 2, were tested for their ability to inhibit the replication of PSR in RC-60 cells (A), BHK-21 cells (0), and RK-13 cells (0). MOI of VSV: BHK-21 cells = 1; RK-13 cells = 45; RC-60 cells = 19. Control yields of PSR (PFUlml): RC-60 = 3.0 x 108; RK-13 = 1.9 x 108; BHK-21 = 4.6 x 108.
VOL. 18, 1976 8 ,^
PSR replication. The RNA+ mutants tsO-23
100
°o _CO
-
6
Cl M
0
E
5
tLA
2 , = A
4
t
3
0
20
531
INHIBITION OF PSR BY VSV B PARTICLES. I.
40
60
80
100
120
UV IRRADIATION (SEC)
FIG. 4. Comparison of the inactiva tion by UV irradiation of infectivity, primary traniscription, and PSR-inhibiting activity of VSV. Aliqzuots of L, VSV were irradiated and tested for infecttivity (A), primary transcription (0), and the inh ibition of PSR replication (@). Primary transcription was measured as follows. Monolayers of BHK-21 cell were infected gs at an MOI of25 (calculated from the ibnfectivity of unirradiated sample). After adsorpt received 2 4 C, each monolayer in 60-mm petri di ml ofHanks balanced salt solution con tamning 5 g of actinomycin D per ml, 100 pg of cyc loheximide per ml, and 5 pCi of [3H]uridine per mil. Infected cultures were harvested after 6 h at 39 C and processed for radioactivity determinations. The iPSR inhibition curve (BHK-21 cells) is taken from IFig. 3. Control yield ofPSR in BHK-21 cells = 4.6 x 1ru u/ml. the
ishes
r
(group Ill) and ts0-45 (group V) interfered with PSR replication to the same extent. Additional representatives of each complementation group will have to be examined before any correlations can be made between the known ts defects and the ability to inhibit PSR replication because of the possible variations in phenotypic expression within the same complementation group (12). DISCUSSION The detection of an activity of a virus preparation that is quantitatively greater than the
concentration of PFU always raises the question of the nature of the virus particle responsible for the activity. Do these virus particles fail to produce plaques because of a defect in the virion or are they victims of chance? At present, this question has not been definitively answered, partly because each virus studied probably presents a different mosaic of these two possibilities. For VSV, the physical particle-toPFU ratio has been reported as 40 to 1 (6) or between 73 to 1 and 194 to 1 (13). It is not known whether the particles that fail to produce plaques are defective. In their study of cell killing by VSV particles, Marcus and Sekellick (11) detected cell killing
particles that were usually in greater concentration than PFU. Although the two activities could not be separated in sucrose gradients, they assumed that cell-killing particles in excess of PFU were defective and referred to them as "defective cell-killing particles." The present report demonstrates that the
number of VSV particles capable of inhibiting the replication of PSR (PSRIP) exceeds the number of PFU by a factor of 32 to 64. This value is in close agreement with the particle-toPFU ratios reported previously (6, 13). The
PSRIP
were
not designated defective because
rr
transcripts from the input genome with an efficiency equal to wild-type virus at 39.5 C. Perhaps this level of RNA synthesis is sufficient to produce inhibition of the replication of PSR. Surprisingly, there was no difference in the inhibition of PSR replication by the RNA- mutant tsO-52 (group II) and the RNA+ mutant tsO23 (group III). Mutant tsO-23, which can carry out primary transcription and replication at 39.5 C, synthesized 60 times more RNA than tsO-52, which shows only primary transcription at the nonpermissive temperature (unpublished observation). Mutant tsG-41, another RNA- mutant that is defective in RNA replication at 39.5 C, had a reduced capacity to inhibit
TABLE 3. Ability of ts mutants of VSV to inhibit the replication of PSR in BHK-21 cells at 39.5 C VSV MU-
V~tanta
PSR control tsO-5 tsG-11 tsG-13 tsG-16
tsO-52 tsO-23 tsG-41 tsO-45 L, VSV a MOI of 5.
Complemenstation
I I I I II III IV V Wild type
RNA synthesis at 39.5
+
+ +
PSyil (PSFU/iel) 1.5 6.1 1.3 1.3 1.1 4.3 1.8 3.8 2.2 2.4
108 104 108 108 108 105 105 107 105 x 104
x x x x x x x x x
532
DUBOVI AND YOUNGNER
there is no direct evidence that this is the case. The high ratio of PSRIP to PFU is not sufficient justification for concluding that the PSRIP are defective. Since there are multiple factors that can influence a PFU assay, the concentration of PFU detected under a particular set of conditions must be considered a relative titer. For example, VSV undergoes an abortive infection in RC-60 cells (15), and a PFU assay of VSV in these cells would be negative. However, a PSRIP assay in RC-60 cells would reveal a high concentration of VSV particles. Were no other cell lines available, one might be inclined to designate all the PSRIP defective. The availability of cell lines more permissive than the RC60 line makes this assumption untenable. Perhaps when more permissive cell lines for VSV are found, the PSRIP-to-PFU ratio will approach 1. At present, we have no direct evidence to suggest that PSRIP and defective cell-killing particles are the same particles. However, it appears unlikely that every inhibitory activity of VSV is due to a unique virus particle. Both PSRIP and defective cell-killing particle activities show a one-particle-per-cell dose response, suggesting that synthesis of a viral product is necessary for inhibition. Also, at the nonpermissive temperature, complementation group I mutants at low MOI are unable to express cell killing (12) or inhibition of PSR replication (Table 3). In addition, group I mutants at high MOI can kill cells (12) and inhibit the replication of PSR (unpublished observation). The UV inactivation kinetics of cell killing (12) and inhibition of PSR replication by 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 replication can range from a 5-log decrease in virus yield to no decrease. The complex UV inactivation curve of PSR inhibition by VSV may reflect different levels of inhibition that depend on the extent of the VSV replication cycle completed by the irradiated particle. For complete inhibition of PSR replication by VSV at low MOI, a fully infectious particle is required for amplification of the input genome to occur. This amplification process exponentially increases the amount of viral products in the cell and results in a rapid increase in the putative inhibitory component that rapidly prevents PSR replication. Without amplification of the input genome, primary transcription can increase viral proteins only in a linear manner, resulting in a slower buildup of the inhibitory components. In this way, synthesis of some PSR virions could occur. The need for amplification to rapidly increase VSV
J. VIROL.
products within the doubly infected cell could explain the high sensitivity of PSR inhibition by VSV to small doses of UV irradiation. The data concerning the PSR-inhibiting activity of UV-irradiated VSV in BHK-21 cells strongly supports the idea of an inhibitory component in the virion of VSV, as has been proposed in numerous reports. In a study of cytotoxicity by UV-irradiated VSV, Cantell et al. (2) showed that UV-irradiated VSV killed cells in the absence of detectable viral-directed protein synthesis. Cell killing required a high MOI of irradiated VSV, and the cytotoxic factor could not be separated from the virus particle. Huang and Wagner (8) demonstrated that UVirradiated VSV inhibited host RNA synthesis in Krebs-2 mouse ascites cells, whereas Yaoi et al. (19) reported a similar phenomenon in CE cells. Marcus and Sekellick (12) suggested that residual primary transcription by VSV may be responsible for the inhibition of host RNA synthesis by UV-irradiated VSV. However, Huang and Wagner (8) employed doses of UV irradiation that would have completely abolished even primary transcription, and their data strongly support the idea of an inhibitory component in the virion of VSV. Additional support for this concept comes from the reports of (i) Wertz and Youngner (17) on the inhibition of host protein synthesis by UV-irradiated VSV, (ii) Yamazaki and Wagner (18) on the cytotoxicity of VSV in interferon-treated cells, and (iii) Huang et al. (7) and Dubovi and Youngner (3) on the inhibition of host RNA synthesis by unirradiated and UV-irradiated DI particles of VSV. An inhibitory component in the virion of VSV may also be responsible for the inhibition of the replication of PSR by inactivated VSV. As with cell killing and the inhibition of host RNA and protein synthesis, inhibition of PSR replication by UV-irradiated VSV requires a high MOI of VSV. The data in Fig. 2 show that approximately 800 to 1,000 times more inactivated particles than infectious particles are necessary to produce equivalent levels of inhibition. In addition, at the nonpermissive temperature, complementation group I mutants, which are unable to inhibit PSR replication at a low MOI of 5 (Table 3), do inhibit PSR replication at a high MOI of 60 (unpublished observation). The inhibition of PSR replication by inactivated VSV and infectious VSV is quantitatively different because of the ability of the infectious particle to amplify the input genome. Flamand and Bishop (4) estimated that a single cell can yield 104 to 105 viral particles. If a protein component of VSV is responsible for the inhibition of PSR replication, it may be necessary to reach a threshold concentration of this
VOL. 18, 1976
INHIBITION OF PSR BY VSV B PARTICLES. I.
protein before interference is expressed. Since heavily irradiated B particles possess little, if any, synthetic activity, it seems likely that a high MOI of irradiated virus would be required to introduce sufficient viral protein to cause inhibition of PSR replication. This situation is somewhat analogous to cell fusion by paramyxoviruses. For fusion-from-without, a high multiplicity of inactivated virus is necessary, but for fusion-from-within, one infectious particle per cell is sufficient. (1). Data presented in an accompanying report (3) concerning the ability of DI particles of VSV to inhibit PSR replication, host RNA synthesis, and host protein synthesis strongly support the concept of an inhibitory component in the virion 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 of VSV, except homologous interference. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant AI-06264 from the National Institute of Allergy and Infectious 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 Press Inc., New York. 2. Cantell, K., Z. Skurska, K. Paucker, and W. Henle. 1962. Quantitative studies on viral interference in suspended L cells. II. Factors affecting interference by UV-irradiated Newcastle disease virus against vesicular stomatitis virus. Virology 17:312-323. 3. Dubovi, E. J., and J. S. Youngner. 1976. Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II. Activity of defective interfering particles. J. Virol. 18:534-541. 4. Plamand, A., and D. H. L. Bishop. 1973. Primary in vivo transcription of vesicular stomatitis virus and temperature-sensitive mutants of five vesicular stomatitis virus complementation groups. J. Virol. 12:1238-1252. 5. Flamand, A., and C. R. Pringle. 1971. The homologies of spontaneous and induced temperature-sensitive mutants of vesicular stomatitis virus isolated in chick
533
embryo and BHK-21 cells. J. Gen. Virol. 11:81-85. 6. Howatson, A. F., and G. F. Whitmore. 1962. The development and structure of vesicular stomatitis virus. Virology 16:466-478. 7. Huang, A. S., J. W. Greenawalt, and R. R. Wagner. 1966. Defective T particles of vesicular stomatitis virus. I. Preparation, morphology, and some biologic properties. Virology 30:161-172. 8. Huang, A. S., and R. R. Wagner. 1965. Inhibition of cellular RNA 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. Radiat. 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 of cell-killing, plaque formation and defective interfering particles of vesicular stomatitis virus. Virology 57:321-338. 12. Marcus, P. I., and M. J. Sekellick. 1975. Cell killing by viruses. II. Cell killing by vesicular stomatitis virus: a requirement for virion-derived transcription. Virology 63;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 and purification of vesicular stomatitis virus by polyethylene glycol precipitation. Virology 40:745-746. 15. Thacore, H. R., and J. S. Youngner. 1975. Abortive infection of a rabbit cornea cell line by vesicular stomatitis virus: conversion to productive infection by superinfection with vaccinia virus. J. Virol. 16:322329. 16. Wertz, G. W., and J. S. Youngner. 1970. Interferon production and inhibition of host synthesis in cells infected with vesicular stomatitis virus. J. Virol. 6:476-484. 17. Wertz, G. W., and J. S. Youngner. 1972. Inhibition of protein synthesis in L cells infected with vesicular stomatitis virus. J. Virol. 9:85-89. 18. Yamazaki, S., and R. R. Wagner. 1970. Action of interferon: kinetics and differential effects on viral functions. J. Virol. 6:421-429. 19. Yaoi, Y., H. Mitsui, and M. Amano. 1970. Effect of UVirradiated vesicular stomatitis virus on nuceleic acid synthesis in chick embryo cells. J. Gen. Virol. 8:165172. 20. Youngner, J. S., H. Thacore, and M. Kelly. 1972. Sensitivity of ribonucleic acid and deoxyribonucleic acid viruses to different species of interferon in cell cultures. J. Virol. 10:171-178.
Vol. 18, No. 2 Printed in U.SA.
Inhibition of Pseudorabies Virus Replication by Vesicular Stomatitis Virus I. Activity of Infectious and Inactivated B Particles EDWARD J. DUBOVI' AND JULIUS S. YOUNGNER* Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received for publication 5 December 1975
Infectious B particles of vesicular stomatitis virus (VSV) are capable of inhibiting the replication of pseudorabies virus (PSR) in a variety of cell lines. Even under conditions of an abortive infection in a continuous line of rabbit cornea cells (RC-60), B particles interfere with the replication of PSR with high efficiency. Particle per cell dose-response analysis of B particle populations revealed that the number of VSV particles capable of inhibiting PSR replication exceeds the number of PFU by a factor of 32 to 64. When B particles are treated with UV irradiation, a drastic increase in the multiplicity of infection is required to inhibit PSR replication. Whereas one infective B particle per cell is sufficient to prevent replication of PSR, 800 to 1,000 VSV particles rendered noninfective by UV irftdiation are required to compensate for the loss of VSV synthetic activity that results from irradiation. Temperature-sensitive mutants representing five complementation groups of VSV were tested at low multiplicities of infection for their effect on PSR replication at the nonpermissive temperature. Generally, the ability of the different complementation groups to amplify virion products at the nonpermissive temperature is associated with their ability to inhibit PSR replication. These results imply that at low multiplicities of infection, amplification of infecting VSV components is necessary for inhibition of PSR replication, but at high multiplicities of infection with VSV, a virion component can prevent PSR replication in the absence of de novo VSV RNA or protein synthesis. Youngner et al. (20) reported that vesicular stomatitis virus (VSV) completely inhibited the replication of pseudorabies virus (PSR) in a continuous line of rabbit kidney cells (RK-13). Rabbit cells in culture are unusual in that pretreatment of such cells with interferon fails to inhibit the replication of DNA viruses, whereas inhibition of RNA viruses is normal (20). Therefore, in RK-13 cells pretreated with interferon, VSV was unable to suppress the replication of PSR. This suggested that the inhibition of PSR replication of VSV is caused by a product synthesized during the VSV replication cycle. The present study reports the conditions necessary for inhibition of PSR replication by VSV in a variety of cell lines. VSV rendered noninfective by UV irradiation and temperature-sensitive (ts) mutants representing five complementation groups of VSV were tested for their ability to inhibit the replication ofPSR.
MATERIALS AND METHODS
Cells. Primary chicken embryo (CE) cells, mouse L cells (clone 929), a line (BHK-21) of hamster kidney cells, and a line (MDCK) of canine kidney cells were propagated in Eagle minimal essential medium plus 4% calf serum. Cell lines of rabbit kidney cells (RK-13), rabbit cornea cells (RC-60), and monkey kidney cells (BSC-1) were grown in MEM plus 10% fetal calf serum. Viruses. The large-plaque mutant of VSVIND (L, VSV) described by Wertz and Youngner (16) was grown in BHK-21 cells. Monolayers in 32-ounce (ca. 960-ml) culture bottles were infected with less than 0.01 PFU per cell to avoid the production of defective interfering (DI) particles. Analysis of [3H]uridinelabeled viral particles by sucrose gradients failed to detect DI particles in lysates produced under these conditions. A single pool of L, VSV was used throughout this study. Virus for this pool was concentrated by polyethylene glycol 6000 as described by McSharry and Benzinger (14) and was partially purified by pelleting the virus through a 50% glycerol cushion. Virus prepared in this manner was stored at -70 C. Temperature-sensitive mutants of VSVIND were obtained from R. R. Wagner. Well-
1 Present address: Department of Microbiology, School of Medicine, University of Virginia, Charlottesville, Va.
22901.
526
VOL. 18, 1976
INHIBITION OF PSR BY VSV B PARTICLES. I.
isolated plaques from terminal dilutions incubated at 32 C were used to produce pools of each mutant in BHK-21 cells at 32 C. The efficiency of plating (39.5/ 32) in CE cells was less than 10-4 for all clones used in this study. PSR originally 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. Cycloheximide was purchased from Upjohn Co., Kalamazoo, Mich., and actinomycin D was obtained through the courtesy of H. B. Woodruff of Merck, Sharpe and Dohme. Double infections with VSV and PSR. Unless otherwise stated, VSV and PSR were added simultaneously to cell cultures in 60-mm petri dishes at an input multiplicity of infection (MOT) of 5 to 10 for each virus. After an adsorption period of 2 h at 37 C in a volume of 1.0 ml, the inoculum was removed by three washes with Eagle minimal essential medium. Three milliliters of growth medium was added, and the cultures were incubated for 18 to 20 h at 37 C. Fluid plus cells were harvested, and the suspension was sonicated to release cell-associated virus. To titrate the PSR, it was necessary to neutralize the infectivity of VSV. This was accomplished by adding anti-VSV antibody to the 10-2 dilution of the virus sample and incubating at 37 C for 1 h. The few VSV plaques that occasionally survived this treatment could easily be discriminated 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 polymerase of VSV was done according to Manders et al. (10). UV-irradiated or unirradiated VSV was added to monolayers of BHK-21 or RC-60 cells at an MOI of 25 (calculated from the unirradiated sample) for 1 h at 4 C. After adsorption, each monolayer, in 60-mm petri dishes, received 2 ml of Hanks balanced salt solution containing 5 ug of actinomycin D per ml, 100 ,ug of cycloheximide per ml, and 5 JLCi of [3H]uridine per ml. After 6 h at 37 C, the acidprecipitable radioactivity per culture was determined as follows. The cell cultures were washed three times with Hanks balanced salt solution and then solubilized with 1% sodium dodecyl sulfate in 0.1 M NaCl and 0.01 M EDTA. Acid-insoluble material was precipitated with 20% trichloroacetic acid, and the precipitates were collected on glass-fiber filters (Whatman GF/A) Inactivation of VSV by UV light. Samples (2.5 ml) of VSV in phosphate-buffered saline were placed in 60-mm glass petri dishes and irradiated with a General Electric 15-W germicidal lamp at a distance of 50 cm. The energy output at this distance was 12 ergs per mm2 as determined by the method of Jagger (9).
RESULTS Inhibition of PSR replication by VSV in different cell lines. Because the inhibition of the replication of PSR by VSV had been demon-
527
strated only in RK-13 cells (20), it was necessary to determine whether this inhibitory activity of VSV was expressed in other cell lines. Various cell lines were infected with VSV and PSR at an MOI of 5 to 10; VSV was added either simultaneously with, or 2 h after, PSR. After 18 h at 37 C, total yields of PSR were determined in CE cells in the presence of anti-VSV antibody. Table 1 shows that in all cell lines tested, VSV completely inhibited the replication of PSR. (Yields of PSR of 104 PFU or less per ml generally represented residual virus remaining after the washing procedure.) The ability of VSV to inhibit the replication of PSR was not directly dependent on the yield of infectious VSV; this can be seen by comparing the yields of VSV in BHK-21 and RC-60 cells. In RC-60 cells, VSV produces an abortive infection that yields less than 1 PFU per cell (15), whereas in BHK-21 cells, VSV replicates to high titers. Even under the conditions of an abortive infection, VSV interfered with the replication of PSR with the same efficiency as in a productive infection. Dose response and estimation of the PSRinhibiting activity of VSV. To estimate the PSR-inhibiting activity of VSV lysates, it was necessary to determine the dose-response curve of the inhibition of PSR replication by VSV. Serial twofold dilutions of the standard VSV preparation were added to monolayers of BHK21 cells simultaneously with PSR (MOI = 5). Three cultures were used for each dilution. Figure 1 shows the average yields of PSR plotted against the number of PFU ofVSV per cell. The solid line is a theoretical one-particle-per-cell dose-response curve generated from the Poisson distribution, whereas the closed circles represent experimentally determined values. Assuming that soluble factors are not responsible for the inhibition of PSR replication, the data in Fig. 1 are paradoxical. At an input MOI of 0.022, more than 98% of the cells would not be infected, and yet the yield of PSR was reduced by over 50%. These data suggest that there are more viral particles 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 mechanism whereby one VSV particle per cell is sufficient to prevent the replication of PSR. Under the assumption that the inhibition of PSR replication by VSV is an all-or-none phenomenon, one can estimate the number of particles in a VSV pool capable of inhibiting PSR replication, since the inhibition process follows
528
J. VIROL.
DUBOVI AND YOUNGNER
TABLE 1. VSV-mediated interference with PSR replication in cells from different species Virus titer (18-h yield)
with VSV (PFU/ml) P Time (h of after superinfection (PFU/ml) PSR infection) VSV VSV (PFUml) PS~a(PFU/ml)
Cell Cell
1.6 x 108
VSV control PSR control
MDCK (canine kidney line)
1.2 x 108 7.7 x 107
0
2
CEM (primary chicken embryo fibroblast)
RK-13 (rabbit kidney line)
L (mouse fibroblast line)
BSC-1 (monkey kidney line)
BHK-21 (baby hamster kidney line)
RC-60 (rabbit cornea line) a
Yields of PSR of less than
VSV control
1.6 x 108
PSR control 0 2
1.4 x 108 1.0 x 108
VSV control PSR control 0 2
2.3 x 106
VSV control PSR control 0 2
8.2 x 107
VSV control PSR control 0 2
1.3 x 108
VSV control
2.6 x 108
PSR control 0 2
2.6 x 108 2.8 x 108
8.6 x 107 1.4 x 108
1.6 x 108 2.8 x 108
(P8SR)
3.4 x 108 4.0 x 104 1.6 x 105
3.9 3.3
4.4 x 107 6.4 x 104 3.5 x 105
2.8 2.1
4.4 x 108 3.2 x 104 3.0 x 103
4.1 5.1
1.2 x 106 3.0 x 103 5.0 x 103
2.6 2.4
9.4 x 107 4.0 x 103 3.0 x 103
4.4 4.5
1.6 x 108 2.0 x 103 7.0 x 103
4.9 4.4
1.2 x 109 7.1 x 103
5.2
1.8 x 105
VSV control PSR control 0
104 represent virus remaining after the washing procedure.
one-particle-per-cell dose response. The titer of PSR-inhibiting particles (PSRIP) was determined using the predictions of the Poisson distribution as described by Marcus and Sekellick (11). If one VSV particle per cell is sufficient to prevent PSR replication, 37% of a population of cells will yield PSR at an MOI of VSV of 1. A virus inoculum that permits a PSR yield of 37% of control values-would contain as many PSRIP as there were cells in the uninfected monolayer. The titer of PSRIP can be determined by knowing the cell number and the dilution of VSV that permits 37% of control yields of PSR. Using this reasoning, the activities of the VSV preparation used to produce the data in Fig. 1 were calculated to be 1.4 x 1010 PSRIP/ml and 3.1 x 108 PFU/ml, a ratio of 45 PSRIP to each PFU. The PSRIP/PFU ratio was also determined with VSV lysates produced in four differa
2.6 x 106 6.5 x 106
og decrease
ent cell lines. Although the yield of PFU varied as much as 100-fold, the PSRIP/PFU ratios were identical in all four lysates, i.e., 32 to 64
by
PSRIP/PFU. Effect of UV irradiation on the PSR-inhibiting activity of VSV. The ability of VSV inactivated by UV irradiation to inhibit PSR replication was dependent on the cell line employed (Table 2). In RK-13 and BSC-1 cells, increasing irradiation caused a loss of ability of VSV to inhibit PSR replication; irradiation of VSV for 90 s reduced the inhibition of PSR yield by only 1 log. In contrast, in BHK-21 cells, the same virus preparation still produced a 4-log drop in PSR yield. The reason for the different efficiency of UV-irradiated VSV in the various cell lines is not clear. It is possible that processing of UV-irradiated VSV influences the inhibition of PSR replication to different degrees in
529 sure of L, VSV to UV light for as long as 300 s still did not change the ability of the irradiated virus to inhibit PSR replication in BHK-21 cells. With VSV given 300 s of irradiation, the PSR yield in doubly infected cells was 1.0 x 103
INHIBITION OF PSR BY VSV B PARTICLES. I.
VOL. 18, 1976
0
z
0 U
0
I-z
10 .
U
a.
0.0
0.022
0.044 0.066 VSV (PFU PER CELL)
0.088
FIG. 1. Dose-response of the inhibition of PSR replication by infective VSV. Serial twofold dilutions of VSV were added simultaneously with PSR (MOI = 5) to monolayers of BHK-21 cells. Infected cultures were harvested at 18 h and assayed for infectivity of PSR in CE cells. The solid line is a theoretical oneparticle-per-cell dose-response curve calculated from the Poisson distribution, and the solid circles represent experimental data. Control yield ofPSR = 3.0 x 108 PFUlml. TABLE 2. Interference with PSR replication: activity of UV-irradiated VSV in different cell lines Exposure 18-h PSR time of VSV (PFU/ Cell line to UV irra- MOI (VSV)a yieldMl) diation (s)
RK-13 0 30 90
20.0 0.0013
0 30 90
26.0 0.0017 0.00017
0.00013
BSC-1
6.0 x 108 1.8 x 105 1.1 x 106
4.1 x 107
2.1 4.3 1.7 1.8
PFU/ml, in contrast to a control PSR yield of 6.0 x 107 PFU/ml. Since UV light primarily damages nucleic acids, these data suggest that an active RNA genome of VSV is not needed for the inhibition of PSR replication in BHK-21 cells and that the VSV virion may contain a component capable of preventing the replication of PSR. If a virion component of VSV is capable of inhibiting the replication of PSR (3), a high MOI of VSV could mask the effect of UV irradiation. To eliminate this possibility, an experiment was designed to assess the ability oflower multiplicities of UV-irradiated VSV to inhibit the replication of PSR. Serial twofold dilutions of unirradiated VSV (control) and VSV irradiated for 360 s were compared for their ability to inhibit PSR replication in BHK-21 cells. (The undiluted, unirradiated control preparation of VSV had an input MOI of 25.) The ability of the irradiated sample to inhibit PSR replication was rapidly lost upon dilution, whereas the unirradiated control still produced significant inhibition at a 10-3 dilution (Fig. 2). Approximately 800 to 1,000 times more irradiated virus was required to achieve the same level of inhi100 0
z
o O 0
UV-IRRADIATED (360 SEC)
10 -
4-CONTROL 1.0
x 10
x 104 x 106 x 107
6.4 x 108 2.8 x 104 17.0 0 3.5 x 104 0.0011 30 4.1 x 104 90 0.00011 a Calculated from the VSV infectivity after exposure to UV irradiation.
BHK-21
RK-13 and BSC-1 cells, on one hand, and in BHK-21 cells, on the other. The ability of UV-irradiated VSV to inhibit PSR replication in BHK-21 cells was examined further using larger doses of UV light. Expo-
0
-I
-2
-3
DILUTION OF VSV
-4
-5
(LOG10)
FIG. 2. Effect of UV irradiation for 360 s on the ability of VSV to inhibit the replication of PSR in BHK-21 cells. Aliquots (2.5 ml) of the standard pool of L, VSV diluted in phosphate-buffered saline were irradiated at a distance of 50 cm using a 15-w General Electric germicidal lamp. Serial twofold dilutions of unirradiated (control) and irradiated VSV were added simultaneously with PSR (MOI = 10) to monolayers of BHK-21 cells. Infected cultures were harvested at 18 h and assayed for infectivity of PSR in CE cells. Control yield of PSR = 1.6 x 108 PFUI ml.
530
DUBOVI AND YOUNGNER
bition produced by unirradiated virus. Whereas one unirradiated VSV particle per cell was sufficient to prevent the replication of PSR (Fig. 1), in the case of irradiated virus, a drastic increase in the MOI was required to compensate for the loss of synthetic activity that resulted from irradiation. Primary transcription by VSV and the inhibition of PSR replication. The effect of UV irradiation on primary transcription by VSV was determined using the in vivo assay of Manders 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 independent of the cell line used; identical results were obtained in BHK-21 cells, a cell 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), we found that VSV infectivity is five times more sensitive to UV irradiation than is primary transcription (slopes of -0.082 and -0.017, respectively; see Fig. 4). To determine the UV inactivation rate of 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 because of the ability of UV-irradiated VSV, at high MOI to inhibit PSR replication in BHK-21 cells (Table 2). Similar experiments were done using RC-60 and RK-13 cells; however, higher MOIs (19 and 45, respectively) were used because these cells are less sensitive than BHK-21 cells to inactivated VSV (Table 2). Virus was irradiated as previously described and tested for its ability to interfere with the replication of PSR. The results (Fig. 3) show that the inactivation 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 in yields of PSR in doubly infected cells.) No direct comparisons can be made between the doses of UV light and the levels of inhibition of PSR replication in these cell lines since the MOI are different. Figure 4 compares the inactivation of the following activities of VSV: infectivity, primary transcription, and inhibition of PSR replication in BHK21 cells. The loss of inhibition of PSR replication by VSV was more sensitive than primary transcription to low doses of UV irradiation. However, VSV irradiated for 110 s still inhibited PSR replication by >90% in BHK-21 cells. Inhibition of PSR replication by ts mutants of VSV at 39.5 C. At least one representative of
J. VIROL.
each of the five complementation groups of VSVIND identified by Flamand and Pringle (5) was tested for its ability to inhibit the replication of PSR at the nonpermissive temperature (39.5 C). The RNA phenotypes at 39.5 C of these mutants agreed with published reports, and the leak and revertant yields of the mutants were less than 0.002 PFU per cell. Monolayers of BHK-21 cells grown in welled trays (16-mm wells) were infected simultaneously with PSR (MOI = 10) and VSV (MOI = 5). After 1 h at 4 C, the inoculum was removed, 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 fluids and cells were havested at 18 h and assayed as previously described. With one exception, complementation group I mutants, which show little or no primary transcription at 39.5 C, did not inhibit the replication of PSR at this temperature (Table 3). Mutant tsO-5 behaved differently than the other group I mutants. Interestingly, Flamand and Bishop (4) reported that tsO-5 produced primary 9
8
E/ 6..
7
U-
0.
0 -J
4
6
0
20
40 60 80 100 UV- IRRADIATION (SEC)
120
140
FIG. 3. Kinetics of inactivation of the PSR-inhibiting activity of VSV in different cell lines. Samples of L, VSV, UV irradiated as described in the legend to Fig. 2, were tested for their ability to inhibit the replication of PSR in RC-60 cells (A), BHK-21 cells (0), and RK-13 cells (0). MOI of VSV: BHK-21 cells = 1; RK-13 cells = 45; RC-60 cells = 19. Control yields of PSR (PFUlml): RC-60 = 3.0 x 108; RK-13 = 1.9 x 108; BHK-21 = 4.6 x 108.
VOL. 18, 1976 8 ,^
PSR replication. The RNA+ mutants tsO-23
100
°o _CO
-
6
Cl M
0
E
5
tLA
2 , = A
4
t
3
0
20
531
INHIBITION OF PSR BY VSV B PARTICLES. I.
40
60
80
100
120
UV IRRADIATION (SEC)
FIG. 4. Comparison of the inactiva tion by UV irradiation of infectivity, primary traniscription, and PSR-inhibiting activity of VSV. Aliqzuots of L, VSV were irradiated and tested for infecttivity (A), primary transcription (0), and the inh ibition of PSR replication (@). Primary transcription was measured as follows. Monolayers of BHK-21 cell were infected gs at an MOI of25 (calculated from the ibnfectivity of unirradiated sample). After adsorpt received 2 4 C, each monolayer in 60-mm petri di ml ofHanks balanced salt solution con tamning 5 g of actinomycin D per ml, 100 pg of cyc loheximide per ml, and 5 pCi of [3H]uridine per mil. Infected cultures were harvested after 6 h at 39 C and processed for radioactivity determinations. The iPSR inhibition curve (BHK-21 cells) is taken from IFig. 3. Control yield ofPSR in BHK-21 cells = 4.6 x 1ru u/ml. the
ishes
r
(group Ill) and ts0-45 (group V) interfered with PSR replication to the same extent. Additional representatives of each complementation group will have to be examined before any correlations can be made between the known ts defects and the ability to inhibit PSR replication because of the possible variations in phenotypic expression within the same complementation group (12). DISCUSSION The detection of an activity of a virus preparation that is quantitatively greater than the
concentration of PFU always raises the question of the nature of the virus particle responsible for the activity. Do these virus particles fail to produce plaques because of a defect in the virion or are they victims of chance? At present, this question has not been definitively answered, partly because each virus studied probably presents a different mosaic of these two possibilities. For VSV, the physical particle-toPFU ratio has been reported as 40 to 1 (6) or between 73 to 1 and 194 to 1 (13). It is not known whether the particles that fail to produce plaques are defective. In their study of cell killing by VSV particles, Marcus and Sekellick (11) detected cell killing
particles that were usually in greater concentration than PFU. Although the two activities could not be separated in sucrose gradients, they assumed that cell-killing particles in excess of PFU were defective and referred to them as "defective cell-killing particles." The present report demonstrates that the
number of VSV particles capable of inhibiting the replication of PSR (PSRIP) exceeds the number of PFU by a factor of 32 to 64. This value is in close agreement with the particle-toPFU ratios reported previously (6, 13). The
PSRIP
were
not designated defective because
rr
transcripts from the input genome with an efficiency equal to wild-type virus at 39.5 C. Perhaps this level of RNA synthesis is sufficient to produce inhibition of the replication of PSR. Surprisingly, there was no difference in the inhibition of PSR replication by the RNA- mutant tsO-52 (group II) and the RNA+ mutant tsO23 (group III). Mutant tsO-23, which can carry out primary transcription and replication at 39.5 C, synthesized 60 times more RNA than tsO-52, which shows only primary transcription at the nonpermissive temperature (unpublished observation). Mutant tsG-41, another RNA- mutant that is defective in RNA replication at 39.5 C, had a reduced capacity to inhibit
TABLE 3. Ability of ts mutants of VSV to inhibit the replication of PSR in BHK-21 cells at 39.5 C VSV MU-
V~tanta
PSR control tsO-5 tsG-11 tsG-13 tsG-16
tsO-52 tsO-23 tsG-41 tsO-45 L, VSV a MOI of 5.
Complemenstation
I I I I II III IV V Wild type
RNA synthesis at 39.5
+
+ +
PSyil (PSFU/iel) 1.5 6.1 1.3 1.3 1.1 4.3 1.8 3.8 2.2 2.4
108 104 108 108 108 105 105 107 105 x 104
x x x x x x x x x
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DUBOVI AND YOUNGNER
there is no direct evidence that this is the case. The high ratio of PSRIP to PFU is not sufficient justification for concluding that the PSRIP are defective. Since there are multiple factors that can influence a PFU assay, the concentration of PFU detected under a particular set of conditions must be considered a relative titer. For example, VSV undergoes an abortive infection in RC-60 cells (15), and a PFU assay of VSV in these cells would be negative. However, a PSRIP assay in RC-60 cells would reveal a high concentration of VSV particles. Were no other cell lines available, one might be inclined to designate all the PSRIP defective. The availability of cell lines more permissive than the RC60 line makes this assumption untenable. Perhaps when more permissive cell lines for VSV are found, the PSRIP-to-PFU ratio will approach 1. At present, we have no direct evidence to suggest that PSRIP and defective cell-killing particles are the same particles. However, it appears unlikely that every inhibitory activity of VSV is due to a unique virus particle. Both PSRIP and defective cell-killing particle activities show a one-particle-per-cell dose response, suggesting that synthesis of a viral product is necessary for inhibition. Also, at the nonpermissive temperature, complementation group I mutants at low MOI are unable to express cell killing (12) or inhibition of PSR replication (Table 3). In addition, group I mutants at high MOI can kill cells (12) and inhibit the replication of PSR (unpublished observation). The UV inactivation kinetics of cell killing (12) and inhibition of PSR replication by 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 replication can range from a 5-log decrease in virus yield to no decrease. The complex UV inactivation curve of PSR inhibition by VSV may reflect different levels of inhibition that depend on the extent of the VSV replication cycle completed by the irradiated particle. For complete inhibition of PSR replication by VSV at low MOI, a fully infectious particle is required for amplification of the input genome to occur. This amplification process exponentially increases the amount of viral products in the cell and results in a rapid increase in the putative inhibitory component that rapidly prevents PSR replication. Without amplification of the input genome, primary transcription can increase viral proteins only in a linear manner, resulting in a slower buildup of the inhibitory components. In this way, synthesis of some PSR virions could occur. The need for amplification to rapidly increase VSV
J. VIROL.
products within the doubly infected cell could explain the high sensitivity of PSR inhibition by VSV to small doses of UV irradiation. The data concerning the PSR-inhibiting activity of UV-irradiated VSV in BHK-21 cells strongly supports the idea of an inhibitory component in the virion of VSV, as has been proposed in numerous reports. In a study of cytotoxicity by UV-irradiated VSV, Cantell et al. (2) showed that UV-irradiated VSV killed cells in the absence of detectable viral-directed protein synthesis. Cell killing required a high MOI of irradiated VSV, and the cytotoxic factor could not be separated from the virus particle. Huang and Wagner (8) demonstrated that UVirradiated VSV inhibited host RNA synthesis in Krebs-2 mouse ascites cells, whereas Yaoi et al. (19) reported a similar phenomenon in CE cells. Marcus and Sekellick (12) suggested that residual primary transcription by VSV may be responsible for the inhibition of host RNA synthesis by UV-irradiated VSV. However, Huang and Wagner (8) employed doses of UV irradiation that would have completely abolished even primary transcription, and their data strongly support the idea of an inhibitory component in the virion of VSV. Additional support for this concept comes from the reports of (i) Wertz and Youngner (17) on the inhibition of host protein synthesis by UV-irradiated VSV, (ii) Yamazaki and Wagner (18) on the cytotoxicity of VSV in interferon-treated cells, and (iii) Huang et al. (7) and Dubovi and Youngner (3) on the inhibition of host RNA synthesis by unirradiated and UV-irradiated DI particles of VSV. An inhibitory component in the virion of VSV may also be responsible for the inhibition of the replication of PSR by inactivated VSV. As with cell killing and the inhibition of host RNA and protein synthesis, inhibition of PSR replication by UV-irradiated VSV requires a high MOI of VSV. The data in Fig. 2 show that approximately 800 to 1,000 times more inactivated particles than infectious particles are necessary to produce equivalent levels of inhibition. In addition, at the nonpermissive temperature, complementation group I mutants, which are unable to inhibit PSR replication at a low MOI of 5 (Table 3), do inhibit PSR replication at a high MOI of 60 (unpublished observation). The inhibition of PSR replication by inactivated VSV and infectious VSV is quantitatively different because of the ability of the infectious particle to amplify the input genome. Flamand and Bishop (4) estimated that a single cell can yield 104 to 105 viral particles. If a protein component of VSV is responsible for the inhibition of PSR replication, it may be necessary to reach a threshold concentration of this
VOL. 18, 1976
INHIBITION OF PSR BY VSV B PARTICLES. I.
protein before interference is expressed. Since heavily irradiated B particles possess little, if any, synthetic activity, it seems likely that a high MOI of irradiated virus would be required to introduce sufficient viral protein to cause inhibition of PSR replication. This situation is somewhat analogous to cell fusion by paramyxoviruses. For fusion-from-without, a high multiplicity of inactivated virus is necessary, but for fusion-from-within, one infectious particle per cell is sufficient. (1). Data presented in an accompanying report (3) concerning the ability of DI particles of VSV to inhibit PSR replication, host RNA synthesis, and host protein synthesis strongly support the concept of an inhibitory component in the virion 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 of VSV, except homologous interference. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant AI-06264 from the National Institute of Allergy and Infectious 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 Press Inc., New York. 2. Cantell, K., Z. Skurska, K. Paucker, and W. Henle. 1962. Quantitative studies on viral interference in suspended L cells. II. Factors affecting interference by UV-irradiated Newcastle disease virus against vesicular stomatitis virus. Virology 17:312-323. 3. Dubovi, E. J., and J. S. Youngner. 1976. Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II. Activity of defective interfering particles. J. Virol. 18:534-541. 4. Plamand, A., and D. H. L. Bishop. 1973. Primary in vivo transcription of vesicular stomatitis virus and temperature-sensitive mutants of five vesicular stomatitis virus complementation groups. J. Virol. 12:1238-1252. 5. Flamand, A., and C. R. Pringle. 1971. The homologies of spontaneous and induced temperature-sensitive mutants of vesicular stomatitis virus isolated in chick
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embryo and BHK-21 cells. J. Gen. Virol. 11:81-85. 6. Howatson, A. F., and G. F. Whitmore. 1962. The development and structure of vesicular stomatitis virus. Virology 16:466-478. 7. Huang, A. S., J. W. Greenawalt, and R. R. Wagner. 1966. Defective T particles of vesicular stomatitis virus. I. Preparation, morphology, and some biologic properties. Virology 30:161-172. 8. Huang, A. S., and R. R. Wagner. 1965. Inhibition of cellular RNA 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. Radiat. 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 of cell-killing, plaque formation and defective interfering particles of vesicular stomatitis virus. Virology 57:321-338. 12. Marcus, P. I., and M. J. Sekellick. 1975. Cell killing by viruses. II. Cell killing by vesicular stomatitis virus: a requirement for virion-derived transcription. Virology 63;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 and purification of vesicular stomatitis virus by polyethylene glycol precipitation. Virology 40:745-746. 15. Thacore, H. R., and J. S. Youngner. 1975. Abortive infection of a rabbit cornea cell line by vesicular stomatitis virus: conversion to productive infection by superinfection with vaccinia virus. J. Virol. 16:322329. 16. Wertz, G. W., and J. S. Youngner. 1970. Interferon production and inhibition of host synthesis in cells infected with vesicular stomatitis virus. J. Virol. 6:476-484. 17. Wertz, G. W., and J. S. Youngner. 1972. Inhibition of protein synthesis in L cells infected with vesicular stomatitis virus. J. Virol. 9:85-89. 18. Yamazaki, S., and R. R. Wagner. 1970. Action of interferon: kinetics and differential effects on viral functions. J. Virol. 6:421-429. 19. Yaoi, Y., H. Mitsui, and M. Amano. 1970. Effect of UVirradiated vesicular stomatitis virus on nuceleic acid synthesis in chick embryo cells. J. Gen. Virol. 8:165172. 20. Youngner, J. S., H. Thacore, and M. Kelly. 1972. Sensitivity of ribonucleic acid and deoxyribonucleic acid viruses to different species of interferon in cell cultures. J. Virol. 10:171-178.
