VIROLOGY
93, 515-526 (1979)
Inhibition
of Vesicular Stomatitis Virus-Defective Interfering Synthesis by Shope Fibroma Virus TIMOTHY R. WINSHIP’
Particle
HARSHAD R. THACORE
AND
Department of Microbiology, School of Medicine, State University of New York at Buffalo, Buffalo, New York 14214 Accepted November
14, 1978
Undiluted passage of vesicular stomatitis virus (VSV) in a line of African green monkey kidney cells results in a cyclic synthesis of standard infectious VSV. This pattern of virus yield is due to the cyclic production of defective interfering (DI) particles [Perrault, J., and Holland, J. J. (1972). Virology 50, 148-158; Palma, B. L., and Huang, A. S. (1974). J. Infect. Die. 129,403-410; Holland, J. J., Villareal, L. P., and Briendl, M. (1976). J. Virol. 17, 805-8131. When such cells were infected with Shope fibroma virus (ShFV) prior to serial undiluted passage of VSV, the normal cyclic yield of VSV was altered. Using two criteria for the determination of DI particles, i.e., the interference assay and the detection of physical particles by gradient technique, it was shown that ShFV exerted its effect by inhibiting the synthesis of VSV-DI particles. It is suggested that ShFV affects both the induction and the amplification of DI particles in this system. Experiments also indicate that the ShFV-mediated inhibition of VSV-DI particle synthesis is probably not due to poxvirus-induced inhibition of cellular macromolecular biosynthesis. INTRODUCTION
et al., 1966;Huang and Wagner, 1966;Huang
It is well documented that in the presence of the drug hydroxyurea, poxviruses are able to rescue vesicular stomatitis virus (VSV) from interferon-induced resistance in various cell lines (Youngner et al., 1972; Thacore and Youngner, 1973a; Thacore, 1976). Furthermore, under similar experimental conditions, VSV replication in some cell lines has been shown to be facilitated by poxviruses in the apparent absence of interferon (Padgett and Walker, 1970; Youngner et al., 1972; Thacore and Youngner, 1973a, 1975; Chen and Crouch, 1978). These observations raise the possibility that the poxvirus not only provides a function(s) for the rescue of VSV from interferon-induced resistance, but may also be affecting other mechanisms which inhibit VSV replication in a given host cell system. One such mechanism operating in VSVinfected cells is the interference phenomenon mediated by defective interfering (DI) particles (Cooper and Bellett, 1959; Huang
and Baltimore, 1970; Huang, 1973). This paper reports the results of experiments conducted in African green monkey kidney (BGM) cells dealing with the synthesis and/or action of VSV-DI particles in the presence of Shope fibroma virus (ShFV). Evidence is presented that during serial undiluted passage of VSV in the presence of ShFV, significantly fewer VSV-DI particles are synthesized than in the absence of ShFV. In contrast, if DI particles are present in large amounts of the VSV inoculum, ShFV does not alter the inhibitory action of DI particles present at the time of infection. Results are also presented which indicate that this inhibition of VSV-DI particle synthesis is probably not due to the general shutdown of host cell macromolecular synthesis induced by the poxvirus. MATERIALS
AND METHODS
Cell cultures. Mouse L cells (clone 929) and primary chicken embryo (CE) cell cul1 To whom requests for reprints should be addressed tures were propagated and maintained in 515
0042~6822/79/040515-12$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
516
WINSHIP
AND THACORE
Eagle’s minimal essential medium (MEM) containing 4% fetal calf serum (Youngner et al., 1966). The African green monkey kidney (BGM) cell line, originated in this laboratory, was grown and maintained in MEM plus 10% fetal calf serum (Barron et al., 1970). Viruses. Two stock preparations of VSV (Indiana strain) were used in this study. One VSV stock was prepared in BGM cells (m.o.i. = 0.001) using an uncloned preparation of VSV. A second VSV stock was prepared in L cells (m.o.i. = 0.001) using a three-times plaque-purified VSV preparation. Low multiplicities of infection were used in the preparation of these two VSV stocks to minimize the production of DI particles. It should be noted, however, that we are unable to detect any DI particle production or decrease in infectious VSV yield during serial undiluted passage of VSV in L cells (unpublished observation). The yield of VSV from infected cells was harvested 48 hr after infection and titrated in primary CE cell monolayers by the plaque method (Youngner et al., 1966). Neither of the two VSV stocks contained detectable amounts of VSV-DI particles. The cytolytic original A strain (ATCC VR-112) of Shope fibroma virus (ShFV), kindly supplied by Dr. J. S. Youngner, was also propagated in BGM cells. Cell cultures were infected with ShFV (m.o.i. = 0.01) and allowed to adsorb for 2 hr at 3’7”. The unadsorbed virus was removed by washing the cultures, refed with medium, and incubated at 3’7” for 72 hr. At the end of this incubation period all of the cells showed cytopathic effects (CPE). The cells were scraped into the culture medium and rapidly frozen and thawed three times to release cell-associated virus. The cell debris was then removed by low-speed centrifugation and the supernatant was assayed for infectivity by the following plaque method. Confluent BGM monolayers in 60-mm petri dishes were infected with 0.5 ml of serial lo-fold dilutions of ShFV and allowed to adsorb for 2 hr at 37”. The monolayers were then washed three times with medium, refed with 3 ml of medium per culture, and reincubated at 37” for 48 hr. Following
this incubation, the culture medium in each dish was removed and the plaques were visualized by staining the monolayers with 0.2% aqueous neutral red. Serial passages of VSV in BGM cell cultures in the presence and absence of ShFV.
Serial undiluted passage of VSV in the presence of ShFV was initiated as follows. Confluent monolayer cultures of BGM cells in 60-mm petri dishes (2.0 x lo6 cells) were first infected with ShFV at an input m.o.i. of 10 and adsorption was carried out for 1.5 hr at 37”. The cell cultures were washed three times with medium to remove unadsorbed virus and then superinfected with VSV at an input m.o.i. of 100. After another adsorption period of 1 hr at 37”, the doubly infected cultures were again washed three times, refed with 3 ml of medium containing 0.05 M hydroxyurea (HU), and reincubated at 37”. This amount of HU was found to inhibit ShFV yield by more than 99% without affecting the replication of VSV. Virus yield was harvested at 48 hr after VSV infection and assayed for VSV infectivity in primary CE cell monolayers. Subsequent passage of this progeny VSV in the presence of ShFV was conducted as described above, using 1 ml of this first-passage stock (Fig. 1). Serial undiluted passages of VSV in the absence of ShFV were also conducted in parallel as described above, except that ShFV was excluded from the protocol (Fig. 1). In order to ensure that the decline in VSV infectivity during the serial undiluted passages was due to the production of DI particles, a series of passages was carried out as follows using diluted inoculum. The VSV yield from each passage series with VSV alone was diluted 1 to 100 prior to infection of BGM cells in the presence and absence of ShFV as described above (Fig. 1). Virus yield was harvested at 48 hr after infection with this diluted VSV and infectivity was determined. The use of 48-hr incubation periods in serial passage experiments was dictated by the finding that under conditions of infection when VSV was present at multiplicities less than or equal to one, the maximum virus yield was obtained onl3; at 48 hr. At higher multiplicities of VSV (10 or more) the maximum yield was ob.
INHIBITION
OF VSV-DI PARTICLE
SYNTHESIS BY ShFV
517
tained between 18 and 24 hr after infection and did not significantly change when harvested at 48 hr (Fig. 2). Detection of VW defective interjiering particles by sucrose density gradient technique. The physical absence or presence
of VSV-DI particles in VSV preparations obtained during serial undiluted passage of VSV in the presence and absence of ShFV was determined by a sucrose gradient technique. Three serial undiluted passages of VSV were carried out according to the protocol shown in Fig. 1, in the presence and absence of ShFV in BGM monolayer cultures grown in 32-0~ bottles (2-4 x lo7 cells/bottle). The culture fluid at each passage was harvested at 48 hr after infection and cell debris was removed by low-speed
Pass0 e Series with Vs V+ShFV
Possa eSeries with V9sVolone
FIG. I. Protocol for passage of VSV in BGM cell cultures in the presence and absence of ShFV. Each circle represents a confluent monolayer (2.0 X lo6 cells). The numbers within the circles represent the passage number. For passage of VSV in the presence of ShFV, the poxvirus was added 1.5 hr prior to infection with VSV. The first VSV passage was carried out at an input m.o.i. of 100. Subsequent undiluted passages were conducted using 1 ml of progeny VSV from the previous passage. All cultures received 0.05 M HU to inhibit ShFV replication. Undiluted passage of VSV alone was also carried out in parallel as described above, except that ShFV was omitted from the protocol. A series of dilute passages in the presence or absence of ShFV representing controls was also conducted by diluting the virus inoculum 1 to 100 prior to infection. Details of the infection orocedures are described in Materials and Methods. Virus yields from all infected cultures were harvested it 48 hr after infection, and VSV infectivity was rssayed in CE cell monolayers.
Hours After Infection
FIG. 2. One-step growth kinetics of VSV in the presence (A - - - A) and absence (0 0) of ShFV in hydroxyurea-treated BGM cells. Monolayers of BGM cells (2 x lo6 cells) were infected with ShFV (m.o.i. = 10) or mock infected with medium and allowed to adsorb for 1.5 hr at 37”. The monolayers were then washed three times with medium, and all cultures were then infected with VSV (m.o.i. = 10). Following a 1-hr absorption period, each monolayer was again washed three times with medium and refed with medium containing 0.05 M hydroxyurea (HU). At various times after infection with VSV, the culture medium was harvested and assayed for VSV infectivity on primary CE cell monolayers as described in Materials and Methods.
centrifugation at 4”. The supernatant fluid was centrifuged at 60,000 g for 2 hr at 4”. The resulting pellet was resuspended in 1 ml of TEN buffer (Holland et al., 1976) and aggregates were dispersed using a Dounce homogenizer. Aliquots (0.5 ml) of this concentrated preparation were layered onto a 15-ml 5 to 40% sucrose density gradient in TEN buffer and centrifuged in a SW 27 rotor at 25,000 rpm for 90 min at 4” (Marcus and Sekellick, 1974). After centrifugation each gradient was obliquely illuminated and the position of visible bands was marked. The contents of each gradient were passed through a fraction collector and 0.5-ml fractions were collected. Each fraction was assayed for infectivity and interfering activity. The empty gradient tubes were used to trace on paper the position of each band. Quantitation
of VSV defective interfering
518
WINSHIP AND THACORE Passage 1
I VSV alone
VSV Sh;"
Passage 2
~~ VSV Olone
vsv Sh;”
Passage 3
irradiated for 60 set using a GE 15-W germicidal lamp from a distance of 50 cm. This uv treatment was found to reduce standard particle infectivity by approximately lOOOfold without affecting DI particle activity (see details in Results; Fig. 3). It should be noted that this uv treatment resulted in the inactivation of significant amounts of standard virus particles. These inactivated particles, however, had no effect on the interfering activity of the DI particles present in the preparation or on the yield of VSV in the assay procedure used. After uv irradiation of each preparation, the residual infectivity was determined by the plaque method. The quantitation of DI particles was conducted in plastic well trays (Bellco Glass, Inc., N. J.). Each well contained a monolayer of 3.5 x lo4 BGM cells. Each monolayer was infected with serial twofold dilutions of uv-irradiated virus stock containing DI particles. Additional VSV (devoid of detectable DI particles) was added to each dilution to ensure a VSV infectious particle m.0.i. of 1. After an adsorption period of 1 hr at 37”, the cells were washed three times with medium, refed with 1 ml of medium, and reincubated at 3’7” for 24 hr. Following this incubation, during which time multiple cycles of VSV replication had occurred, the culture medium was harvested from each well and assayed for infectivity in CE cell monolayers as described above. The infectious VSV yield from each well infected with standard and DI particles was compared with the yield from wells infected with standard infectious VSV alone (control). The percentage inhibition of VSV yield due to DI particles at each dilution was calculated. This was plotted on a semilogarithmic graph against the dilution of the sample containing DI particles. From the linear, one-hit curve obtained, the sample dilution inhibiting the VSV yield by 63% was calculated. It should be noted that the lack of one-hit kinetics observed by Bellett and Cooper (1959) under certain conditions was not encountered under our experimental conditions. From such a one-hit curve, it can be inferred that the number of DI particles is equal to the number of
IIC VSV alone
vsv
sh
FIG. 3. Detection of VSV defective interfering particles by sucrose density gradient centrifugation. BGM monolayers (2-4 x 10’ cells) in 32-0~ bottles were used for serial undiluted passage of VSV in the presence and absence of ShFV according to the protocol shown in Fig. 1. The virus yield from each passage was concentrated by centrifugation at 65,000g for 2 hr as described in Materials and Methods. The viral concentrate from each passage was layered onto a 5 to 40% sucrose gradient in TEN buffer, and centrifugation was carried out for 90 min at 25,000 rpm in an SW 27 rotor at 4” (Marcus and Sekellick, 1974). Following centrifugation, bands were visualized by the use of obliquely transmitted light and the position of each band was marked on the tube. The gradients were then fractionated as described in the text and each fraction was analyzed for PFU and interfering activity. The lowest band present in each gradient marks the position of VSV-“B” particles, as confirmed by the presence of maximum infectivity and the lack of detectable interfering activity. The two additional bands present near the middle of the gradient (VSV alone, passage 2) represent DI-particle bands, as confirmed by the presence of interfering activity. The VSV yields (PFU/ml) in passage 1 were 2.4 x 1OB (VSV alone) and 6.5 x lo8 (VSV + ShFV); passage 2, 8.2 x lo8 (VSV alone) and 8.5 x lOa (VSV + ShFV); and passage 3, 7.0 x lo* (VSV alone) and 1.6 x lo8 (VSV + ShFV).
particles. DI particles were quantitated using a modified version of the biological assay of Bellett and Cooper (1959). It was noted that their original procedure was not very sensitive if the preparation containing DI particles also contained large numbers of standard infectious particles. This could be overcome by uv irradiation of such preparations (Fig. 3) since infectious particles of VSV are significantly more sensitive to uv irradiation than DI particles (Holland et al., 1976). For this reason, prior to the quantitation of DI particles by this yield reduction method, all samples were uv
INHIBITION
OF VSV-DI
PARTICLE
cells per culture. Since this cannot be directly confirmed, we have chosen to express the interfering activity as interfering units rather than DI particles. An interfering unit is defined as the number of DI particles necessary to inhibit the VSV yield by 63% multiplied by the reciprocal of the number of cells per culture. Effect of ShFV on BGM cellular RNA and protein synthesis. Confluent monolayers (2.0 x lo6 cells) were infected with ShFV at an input m.o.i. of 10 in the usual manner. After adsorption, cultures were washed, refed with medium alone or with medium containing 0.05 M HU, and reincubated at 37”. At various times after infection, cultures were labeled with either [3H]uridine (1 &X/ml) or [3H]leucine (1 &i/ml) for 30 and 20 min, respectively. After the labeling period, the monolayers were washed and acid-insoluble radioactivity was determined as previously described (Thacore and Youngner, 1972). Chemicals. [3H]Uridine (specific activity, 25 Wmmol) and [3H]leucine (specific activity, 61 CWmmol) were purchased from Schwarz/Mann, Orangeburgh, N. Y. Hydroxyurea, A grade, was obtained from Calbiochem, Calif. RESULTS
One-step growth kinetics of VW in the presence and absence of ShFV in hydroxyurea-treated BGM cell cultures. Preliminary experiments were conducted to determine the effect of ShFV on VSV replication in BGM cells. A one-step growth curve of VSV was conducted in the presence and absence of ShFV as described in the legend to Fig. 2. It should be noted that 0.05 M hydroxyurea (HU) was added to cultures to inhibit the replication of ShFV without affecting the VSV yield (Youngner et al., 1972; Thacore, 1976). At various times after infection, culture fluid was harvested and assayed for progeny VSV by plaque assay on primary CE cell monolayers. The presence of HU allowed us to assay for VSV in the absence of any significant amount of the poxvirus. The results presented in Fig. 2 show that no significant difference
SYNTHESIS
BY ShFV
519
was observed either in the growth curve or in the total VSV yield per cell in the presence or absence of ShFV, indicating that in BGM cells, the VSV yield was not facilitated by ShFV. The maximum VSV yield was obtained at 24 hr after infection, but this level of infectivity was maintained for up to 48 hr after infection. These results suggest that ShFV does not affect the attachment, penetration, or replication of VSV under these experimental conditions. VSV yield during undiluted passage in the presence and absence of ShFV in BGM cell cultures. To study the effect of ShFV on the VSV yield during undiluted serial passage in BGM cells, experiments were conducted according to the protocol shown in Fig. 1. VSV pools prepared from cloned and uncloned VSV in L and BGM cells, respectively, were used in these experiments. An initial m.o.i. of 100 was used for passage 1, and subsequent passages were made using 1 ml of undiluted virus progeny from the previous passage. As controls, virus yield from undiluted passage in the absence of ShFV was diluted 1 to 100 in medium prior to infection, represented by the dilute passage series in Fig. 1. VSV yields obtained during four such serial passages are shown in Table 1. When uncloned VSV (prepared in BGM cells) was passaged undiluted in the absence of ShFV, the yield of infectious VSV dropped markedly, from 1.3 x lOa PFU/ml in passage 1 to 9.2 x lo4 PFU/ml in passage 4 (group 2). Similar results were obtained with cloned VSV prepared in L cells (group 6). The drastic drop in virus yield during undiluted passage obtained with either cloned or uncloned VSV was prevented when the virus was diluted 1 to 100 prior to passage (groups 1 and 5). This suggested that (a) the decrease in VSV yield during undiluted passage was due to the production of DI particles in the system, and (b) normal VSV yield could be obtained if the virus preparation was diluted to reduce DI particle numbers in the preparation prior to infection. Similar results have been obtained with VSV in other cell systems (Perrault and Holland, 1972; Palma and Huang, 1974; Holland et al., 1976; Kang et al., 1978).
520
WINSHIP AND THACORE TABLE 1
VSV YIELD DURING SERIAL PASSAGEIN THE PRESENCEAND IN THE ABSENCE OF ShFV IN BGM CELLS Virus infection
VSV yield (PFU/ml) Passage number
Source of VSV preparation
VSV
ShFV
Method of passage of vsv
Uncloned in BGM cells
+ +
-
Diluted” Undiluted
1 2
+ +
+ +
Diluted Undiluted
3 4
+ +
-
Diluted Undiluted
5 6
+ +
+ +
Diluted Undiluted
7 8
Cloned in L cells
Experimental group
1
2
3
4
3.5 x 108 1.3 x lOa (0.4)b 1.7 x lo* 2.0 x 108 (0.0)
1.3 x 108 2.3 x lOs (0.0) 1.9 x lo* 1.8 x lo8 (0.0)
3.4 x 108 5.8 x lo6 (1.7) 1.7 x lo8 7.2 x 10’ (0.4)
1.0 x 108 9.2 x lo4 (3.3) 4.2 x lo8 2.3 x 10’ (1.3)
2.6 x lOa 1.8 x lo* 1.9 x 10s 1.8 x lOa (0.0) (0.2) 1.9 x 108 1.2 x 108 1.2 x 108 2.0 x 108 (0.2) (0.0)
1.8 x 10” 6.6 x lOa 1.5 x lo6 2.8 x lo5 (2.5) (3.1) 1.4 x 108 1.1 x 108 2.0 x 10s 2.1 x 10’ (0.2) (0.9)
a Undiluted virus preparation was diluted 1 to 100 prior to infection. * Log drop in virus yield as compared to dilute passage yield.
In contrast to the results obtained above, when cloned or uncloned VSV was passaged undiluted in the presence of ShFV, the drastic drop in VSV yield observed in the absence of the poxvirus was strikingly absent. In passage 4, for example, the yield of uncloned VSV dropped by 1.3 and 3.3 log in the presence and absence of ShFV, respectively (compare group 2 with group 4). Similarly, with plaque-purified VSV, during passage 4, the infectious virus yield dropped by 0.9 and 3.1 log in the presence and absence of ShFV, respectively (compare groups 6 and 8). It should be noted that the yield of infectious VSV from dilute passages with cloned and uncloned VSV did not vary significantly, in the presence or absence of ShFV, during the four serial passages (compare group 1 with group 3, and group 5 with group 7). These results indicate that, under these experimental conditions, ShFV presented the marked drop in VSV yield during serial undiluted passages of VSV in BGM cultures to a great extent. To differentiate our results from those of Kang and Allen (1978), further experiments dealing with the effect of ShFV on DI particle synthesis and/or action were
conducted using the uncloned, BGM-grown preparation of VSV. Effect of ShZW on interference mediated by DZ particles. The question arose: Could the decline in VSV yield observed during serial undiluted VSV passages in the absence of ShFV (Table 1, group 2, passages 3 and 4) be prevented by the addition of ShFV to the inocula of these passages, rather than at the beginning of the passage series? To answer this question, virus stock from undiluted passage 3 (Table 1, group 2) was again passaged undiluted, in both the presence and the absence of ShFV, in the usual manner. The virus yield was harvested at 48 hr after infection and assayed for infectivity. The results, presented in Table 2, show that the presence of ShFV failed to prevent a sharp decline in infectious VSV yield. There was no significant difference in the VSV yield from that observed when this VSV stock was passaged in the absence of ShFV (compare Table 1, group 2, passage 4, with Table 2, groups 2 and 4). It would therefore appear that the effect of ShFV observed in Table 1 is to prevent the buildup, from passage 1, of some factor(s), probably active DI particles, neces
INHIBITION
OF VW-D1 PARTICLE
sary for the decrease in infectious VSV yield. Recently, strong evidence has been presented that the mechanism of DI-mediated interference with the replication of standard infectious VSV and the replication of DI particles themselves are separate events (Khan and Lazzarini, 19’7’7;Adachi and Lazzarini, 19’78).The data presented in Tables 1 and 2 indicate that ShFV may exert some effect on the synthesis of active VSV-DI particles and not on the ability of these particles to interfere with the replication of standard infectious particles. The interference observed in Table 2 is most likely due to the presence of DI particles in the virus inoculum (yield from passage 3). If ShFV affects the synthesis of VSV-DI particles, two modes of action are possible. First, ShFV could reduce the numbers of DI particles synthesized. Second, ShFV infection could result in the synthesis of altered subviral particles physically similar to DI particles but unable to interfere with standard VSV replication. To help distinguish between these two possibilities, experiments were conducted to demonstrate the physical presence or absence of VSV-DI particles in the presence of ShFV. Detection of VSV defective interfering particles by sucrose density gradient technique . Undiluted passages of VSV were
conducted in the presence and absence of ShFV according to the protocol shown in Fig. 1. The experimental conditions are described in detail in the legend to Fig. 3 and in Materials and Methods. The virus yields from three serial undiluted passages were clarified and concentrated as described in Materials and Methods. The concentrated preparations were placed onto 5-40% sucrose density gradients and centrifuged in a SW 27 rotor at 25,000 rpm for 90 min at 4”. The bands were visualized as described in Materials and Methods, and the results obtained are shown in Fig. 3. In the tist undiluted passage, only one visible band was observed, in both the presence and the absence of ShFV. These bands, in both the gradients, were confirmed to be “B’‘-partitle bands by the presence of maximum infectivity and the lack of detectable inter-
SYNTHESIS BY ShFV
521
TABLE 2 EFFECT OF ShFV ON VSV YIELD IN BGM CELLS INFECTED WITH THE THIRD SERIAL UNDILUTED PASSAGE VSV STOCK
Method of infection with VSV stock”
ShFV (m.0.i. = 10)
Experimental group
VSV yield (PFU/ml)
Dilutedb Undiluted
-
1 2
2.5 x lo* 3.0 x 105
Diluted Undiluted
+ +
3 4
5.2 x lo8 1.8 x lo5
” This stock represents the third serial undiluted VSV passage yield in the absence of ShFV (Table 1, passage 3, group 2). This preparation contained 5.8 x lo6 PFU/ml. * Virus stock was diluted 1 to 100 prior to infection,
fering activity. In the second serial undiluted VSV passage without ShFV, two additional bands were observed in the gradient above the “B’‘-particle band. High interfering activity was associated with these two bands, which were therefore confirmed to be DI particles (Fig. 3). In contrast, in the same passage in the presence of ShFV, no visible DI bands were observed in the gradient (Fig. 3). In the third serial undiluted passage of VSV in the absence of ShFV, no visible bands were observed, probably due to the strong interference mediated by the DI particles present in the previous passage. However, in the same passage in the presence of ShFV, a visible “B”-particle band was noted. No DIparticle bands were present in the third passage in the presence of ShFV. These results strongly suggest that the synthesis of both species of DI particles was significantly reduced when VSV was serially passaged undiluted in the presence of ShFV. It should be emphasized that the detection of DI bands in a sucrose density gradient can only be considered a qualitative measure of DI particles. To confirm the above findings (Fig. 3) and to further quantitate the number of DI particles at each passage level in the presence and absence of ShFV, the following experiments were conducted.
522
WINSHIP AND THACORE
0
60
ix)
180
Secondsof UV-Irradiation FIG. 4. Inactivation of VSV-DI and VSV infectious particle activity by uv irradiation. A VSV stock containing 8.0 x 10’ PFU/ml and 2.0 x 10B interfering units/ml was uv irradiated using a GE l&W germicidal lamp from a distance of 50 cm. After uv irradiation for various periods of time, the residual infectivity (PFU) (0 0) and the DI-particle activity (interfering units) (0 - - - 0) were determined as described in Materials and Methods.
Quantitution of VW defective interjking particles. We made use of an assay system which took into account one of the most important properties of DI particles; that is, the ability of these particles to interfere with the replication of standard VSV particles. Preliminary experiments were conducted to devise a procedure to detect relatively small numbers of biologically active interfering particles in a preparation containing large numbers of infectious particles, as would be necessary when using a virus stock that had been passaged undiluted once or twice in BGM cells. Advantage was taken of the fact that the DI particles of VSV and lymphocytic choriomeningitis (LCM) virus have been found to be less sensitive to uv irradiation than infectious particles (Welsh et al., 1972; Holland et al., 1976). Attempts were therefore made to reduce the infectious VSV titer by uv irradiation, without affecting the DI particle
interfering ability, to an extent which would allow the detection of relatively small numbers of biologically active VSV-DI particles using the procedure of Bellett and Cooper (1959). A VSV stock containing 8.0 x 10’ PFU/ml and 2.0 x lo8 DI particles/ml (as determined by the unmodified Bellett and Cooper procedure) was uv irradiated for various periods of time. The residual infectivity and DI particles were quantitated as described in detail in Materials and Methods. The data presented in Fig. 4 show that uv irradiation of such a VSV stock preparation for 60 set resulted in a lOOO-foldreduction in infectivity without affecting the biological activity of the DI particles present. Ultraviolet irradiation for more than 60 set results in a decrease in DI particle activity. It should be emphasized that even though uv irradiation for 60 set results in the formation of large numbers of uv-inactivated particles in the preparation, these inactivated particles do not significantly affect the interfering ability of the DI particles present in the preparation, nor do they affect the replication of the VSV “B” particles in the assay system employed (Winship and Thacore, unpublished observation). The results obtained in Fig. 4 are similar to those obtained for LCM virus (Welsh et al., 1972), but differ somewhat from those obtained for VSV-DI particles by Holland et aZ. (1976). The discrepancy between Holland’s data and our own (Fig. 4) could be due to the assay procedure used to quantitate DI particles. Holland’s group made use of an assay for DI particles that was dependent on DI particle replication, while the assay used in this study and the procedure used by Welsh et al. depended on the ability of DI particles to interfere with VSV replication. Differences in the sensitivity of these two DI particle functions to uv irradiation may account for the differences between our data and those reported by Holland’s group. The relative insensitivity of the interfering ability of VSV-DI particles to 60 set of uv irradiation (under the conditions described in Fig. 4) allowed us to detect as few as 3.5 x lo4 DI particles/ml in a preparation containing lOOO-foldmore infectious
INHIBITION
OF VSV-DI PARTICLE
virus than DI particles. This lower limit of detection of DI particles is significantly more sensitive than other procedures such as inhibition of virus-specific RNA synthesis (Huang, 1977) and nonbiological methods such as banding in sucrose density gradients (Holland et al., 19’76). In an attempt to quantitate the number of DI particles synthesized during undiluted passage of VSV in BGM cells in the presence and absence of ShFV, experiments were conducted as described above according to the protocol in Fig. 1. Eight serial undiluted passages of VSV were carried out in the presence and absence of ShFV, and the infectious virus and DI particle yields at each passage were determined. The results are shown in Fig. 5. Undiluted serial passage of VSV in the absence of ShFV resulted in a cyclic pattern of synthesis of infectious virus, as had been noted by other workers (Fig. 5a). In the presence of ShFV the cyclic pattern of infectious virus synthesis was altered (Fig. 5b). Moreover, approximately IOO-foldmore infectious virus was synthesized in the presence of ShFV than in its absence, especially in passages4 and 7. As expected, a cyclic pattern of DI particle synthesis was obtained in the undiluted VSV passage series in the absence of ShFV (Fig. 5a). Only in passage 5, which contained 6.0 x lOa PFU/ml, was no interfering activity detected. In contrast, in the presence of ShFV, except for passages 3 and 4, no interfering activity was detected (Fig. 5b). It is interesting to note that no interfering activity was detected during the second cycle of DI particle production (passages 5 through 8) in the presence of ShFV, whereas significant amounts of interfering units were detected in the absence of the poxvirus. It should also be noted that the yield of VSV from diluted passages in the presence and absence of ShFV varied only twofold, or less, and the titers ranged from 6 x lo8 to2 x 10gPFU/ml(datanot shown). These results clearly demonstrate that ShFV prevented the synthesis of DI particles during serial undiluted passagesof VSV in these cells. The results presented above show that ShFV prevents some aspect of VSV-DI
SYNTHESIS BY ShFV
523 b
I I
L
2
3
4
5
6
7
8
1
2345670
Number Of Undiluted Serial Passages
FIG. 5. Yield of infectious VSV and interfering units during undiluted passages of VSV in BGM cells in the absence (a) and in the presence (b) of ShFV. VSV was passaged undiluted in the presence and absence of ShFV according to the protocol shown in Fig. 1. Details of the experimental procedures are described in the legend to Fig. 1 and in Materials and Methods. Virus yields were harvested at 48 hr after infection and assayed for PFU (0 0) on CE cell monolayers and for interfering units (0 - - - 0) by the modified Bellett and Cooper (1959) assay procedure described in Materials and Methods.
particle synthesis and not the ability of VSV-DI particles to interfere with the replication of infectious VSV particles. Several modes of ShFV action on VSV-DI particle synthesis can be postulated. First, some ShFV-directed product may interact directly with the replicating VSV genome so as to prevent the synthesis of DI particles; second, a ShFV-directed product(s) could prevent the action of some preexisting host cell factor necessary for VSV-DI particle synthesis; and third, ShFV could prevent the synthesis of a host cell function necessary for the synthesis of VSV-DI particles by inhibiting host (RNA and protein) synthesis (Rang and Allen, 1978). Of these three possibilities, the third
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0.05 M HU. At various times after infection, the cultures were labeled with [3H]uridine or [3H]leucine in the presence or absence of HU as described in Materials and Methods. After an appropriate labeling period, the cellular acid-insoluble radioactivity was determined as described previously (Thacore and Youngner, 1972). The results are shown in Fig. 6. In cultures not treated with HU, at 8 hr after infection, cellular RNA and protein syntheses were 75 and 54% those of uninfected control cultures, respectively. However, in the presence of HU, the inhibition of cellular RNA and protein synthesis was less marked. In such cultures, at 8 hr after infection, cellular RNA and protein syntheses were 83 and 20 75% those of uninfected control cultures, respectively (Fig. 6). These results suggest 10 that the indiscriminate inhibition of cellular RNA and protein synthesis by ShFV does 1 01 not appear to be the mechanism by which 6 7 4 5 2 3 8 f the poxvirus inhibits the synthesis of VSV-DI Hours After ShFV lnfectm particles during undiluted serial passages FIG. 6. Effect of ShFV on BGM cellular RNA and in BGM cell cultures.
protein synthesis. Monolayer cultures were infected with ShFV (m.o.i. = 10) in the usual manner. After infection, the cultures were refed with medium alone or with medium containing 0.05 M HU. At designated intervals after infection, the rates of RNA (A --- A) and protein (0 0) synthesis in cultures treated with medium alone and RNA (A - - - A) and protein (0 0) synthesis in cultures treated with HU were measured as described in Materials and Methods. Uninfected cultures were used as controls. The average incorporations of radioactivity by uninfected cultures treated with medium alone were 86,766 cpm ([3H]uridine) and 93,002 cpm ([3H]leucine); those by cultures treated with HU were 85,066 cpm ([3H]uridine) and 89,666 cpm ([sH]leucine).
DISCUSSION
The undiluted passage of VSV in the BGM line of African green monkey kidney cells resulted in a cyclic pattern of infectious VSV yield due to the production of DI particles. However, when BGM cells were infected with ShFV prior to the undiluted passage of VSV, the cyclic pattern was altered (Fig. 5). This effect was due to the reduced synthesis of DI particles, as confirmed by the absence of DI-particle bands during three serial undiluted passages and by the quantitation of biologically active DI particles during eight serial undiluted seemed the most likely and the most acces- passages in the presence and absence of sible to experimental verification. Experi- ShFV. The quantitation of DI particles ments were therefore conducted to study was conducted using a procedure increasthe effect of ShFV on cellular RNA and ing the sensitivity of the biological interprotein synthesis. ference method of Bellett and Cooper (1959). Effect of ShFV on BGM cellular RNA The modified procedure involved the difand protein synthesis. BGM monolayers ferential effect of uv light on DI and infec(2.0 x 10” cells) were infected with ShFV tious VSV particles, as has been previously (m.o.i. = 10) in the usual manner. After noted for VSV by Holland et al. (1976) and adsorption, the cultures were refed with for LCM virus by Welsh et al. (1972). This medium alone or with medium containing uv treatment, along with relatively small
INHIBITION
OF VSV-DI PARTICLE
numbers of cells per monolayer culture, increased the sensitivity of the test and allowed the detection of as few as 3.5 x lo4 DI particles/ml in a preparation containing large amounts of infectious virus. A cyclic pattern of DI particle synthesis was also observed during undiluted passage of VSV in the absence of ShFV. The synthesis of the DI particles, however, lagged behind by one passage as compared to the cyclic yield of infectious VSV. Such a cyclic relationship between DI and infectious particles has been postulated by Huang and her coworkers (Huang, 1973; Palma and Huang, 1974) and observed by Popescu et al. (1976). It is interesting to note that during serial undiluted passages of VSV, the infectious virus yield in a given passagedepends largely on the amount of DI particles synthesized in the previous passage, rather than on the number of DI particles synthesized during that given passage (Fig. 5a). To speculate on the mode of action of ShFV on DI particle synthesis would be premature at this time, since very little is known regarding the synthesis of DI particles. There seem to be, however, two stages in DI particle synthesis. The first involves the induction of a small number of DI particles, a process in which host cell functions may play an important role (Kang and Allen, 1978). The second stage involves the replication or amplification of the small number of DI particles induced in the first stage. Experiments are in progress to determine conclusively whether ShFV acts at the level of DI particle induction and/or amplification. The results presented in Table 1, however, with cloned VSV in L cells (groups 5 to 8) shed some light on this question. As mentioned previously, we have been unable to detect either the production of VW-D1 particles or a decrease in VSV yield during serial undiluted passage of cloned or uncloned VSV in L cells. It should be noted that strong interference with VSV replication is observed in these cells when VSV-DI particles are added (unpublished observation). These results suggest that VSV stocks prepared in L cells are devoid of DI particles. When such a VSV stock is used for serial undiluted pas-
SYNTHESIS BY ShFV
525
sages in BGM cells, the decrease in VSV yield due to the production of DI particles was largely prevented by ShFV, indicating that ShFV must reduce the induction of DI particles in BGM cells. A strong possibility also exists that the amplification of DI particles is affected by ShFV in these cells. At this time, however, we have no direct evidence for this except that the effect of ShFV is observed with both cloned and uncloned VSV stocks (Table 1). This observation differs from that reported by Kang and Allen (1978). This could best be explained if the amplification of VSV-DI particles were also being affected by ShFV. The exact biological function(s) supplied by ShFV is unknown at this time. It is evident, however, that ShFV-mediated inhibition of DI particle synthesis takes place in the presence of HU, indicating that ShFVDNA synthesis is not required. It is interesting that the ability of poxviruses to rescue VSV from interferon-induced resistance and to facilitate VSV yield in the absence of interferon is also resistant to HU (Thacore and Youngner, 1973b; Thacore, 1976; Chen and Crouch, 1978). The possibility that a particular poxvirus function may be responsible for all three activities cannot be ruled out at this time. ACKNOWLEDGMENT This work was supported by Public Health Service Grant 50-E 129D. REFERENCES ADACHI, T., and LAZZARINI, R. A. (19’78).Elementary aspects of autointerference and the replication of defective interfering particles. Virology 87, 152-163. BARRON, A. L., OLSHEVSKY,C., and COHEN, M. M. (1970). Characteristics of the BGM line of cells from African green monkey kidney. Arch. Virusforsch. 32, 389-392. BELLETT, A. J. D., and COOPER,P. D. (1959). Some properties of the transmissable interfering component of vesicular stomatitis virus preparations. J. Gen. Wcrobiol. 21, 498-501. CHEN, C. Y., and CROUCH,N. A. (1978).Shope fibroma virus-induced facilitation of vesicular stomatitis virus adsorption and replication in nonpermissive cells. Virology 85, 43-62. COOPER,P. D., and BELLETT, A. J. D. (1959). A transmissable interfering component of vesicular
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Holland, J. J., VILLAREAL, L. P., and BRIENDL, M. (1976). Factors involved in the generation and replication of rhabdovirus defective T particles. J. Viral.
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HUANG, A. S., GREENAWALT, J. W., and WAGNER, R. R. (1966). Defective T particles of vesicular stomatitis virus. I. Preparation, morphology, and some biologic properties. Virology 30, 161-172. HUANG, A. S., and WAGNER, R. R. (1966). Defective T particles of vesicular stomatitis virus. II. Biologic role in homologous interference. Virology 30, 173- 181. KHAN, S. R., and LAZZARINI, R. A. (1977). The relationship between autointerference and the replication of a defective interfering particle. Virology 77, 189-201. KANG, C. Y., and ALLEN, R. (1978). Host-function dependent induction of defective interfering particles of vesicular stomatitis virus. J. Viral. 25, 202-206. KANG, C. Y., GLIMP, T., CLEWLEY, J. P., and BISHOP, D. H. L. (1978). Studies on the generation of vesicular stomatitis virus (Indiana serotype) defective interfering particles. Virology 81, 142- 152. MARCUS, P. I., and SEKELLICK, M. J. (1974). Cell killing by viruses. I. Comparison of cell-killing, plaque-forming, and defective-interfering particles of vesicular stomatitis virus. Virology 57,321-338. PADGETT, B. L., and WALKER, D. L. (1970). Effect of persistent fibroma virus infection on susceptibility of cells to other viruses. J. Viral. 5, 199-204. PALMA, B. L., and HUANG, A. S. (1974). Cyclic production of vesicular stomatitis virus caused by defective interfering particles. J. Infect. Dis. 129, 403-410.
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WELSH, R. M., O’CONNELL, C. M., and PFAU, C. J. (1972). Properties of defective lymphocytic choriomeningitis virus. J. Gelz. Viral. 17, 355-359. YOUNGNER, J. S., SCOTT, A. W., HALLUM, J. V., and STINEBRING, W. R. (1966). Interferon production by inactivated Newcastle disease virus in cell cultures and in mice. J. Bacterial. 92, 862-868. YOUNGNER,J. S., THACORE,H. R., and KELLY, M. E. (1972). Sensitivity of ribonucleic acid and deoxyribonucleic acid viruses to different species of interferon in cell cultures. J. Viral. 10, 171-178.