- Email: [email protected]
Infectious reovirus subviral particles: Virus replication, cellular cytopathology, and DNA synthesis
VIROLOGY
74, 259-261
Infectious
(1976)
Reovirus Subviral Cytopathology, DONALD
Department
of Botany
C. COX’ and
Particles: Virus Replication, and DNA Synthesis’ C. WORTH
AND
Microbiology,
University
Accepted
July
Cellular
CLINKSCALES”
of Oklahoma,
Norman,
Oklahoma
73069
14,1976
The formation of virions in cells infected with reovirus infectious subviral particles (SVPi) appears to begin earlier, proceeds at a greater rate, and results in a threefold higher yield of progeny virus than in infections with complete virions. However, SVPi infection results in significantly reduced nonspecific cytotoxic effects. When corrections are made for cytotoxic effects, SVPi inhibit cell DNA synthesis at the same rate and to the same degree as intact reovirus.
Reovirus infection results in an inhibition of cell DNA replication (1+2). This inhibition appears to be a consequence of the prevention of the initiation of DNA synthesis (3,4) and is not related to modifications in precursor pools, DNA polymerase activity, or template integrity (5,6). An investigation of DNA synthesis in cells treated with specific subviral particles which are missing certain virion subunits could identify the viral components or replicative functions which mediate the inhibition of cell DNA synthesis. Recent studies (7,8) have shown that two noninfectious reovirus subviral particles, top component and cores, do not cause such inhibition. Reovirus subviral particles produced by limited chymotrypsin digestion lack certain capsid polypeptides but retain infectivity (9, 10). The experiments described in this report compare virus replication characteristics, modification of cell DNA synthesis, and the development of nonspecific cytotoxicity in cells treated I Portions of this work were presented at the meeting of the American Society for Microbiology, 1974, Chicago, Illinois. * Author to whom requests for reprints should be addressed. 3 Present address: Microbiology Department, University of Massachusetts Medical School, Worcester, Mass. 259 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
with complete reovirus and infectious subviral particles (SVPi). SVPi adsorb to cells as efficiently as intact reovirus (unpublished). It was necessary to determine whether this interaction was reflected in an equally efficient virus replication cycle. Significant levels of infectious virus are detectable earlier, the rate of progeny production appears to be greater, and the final yield of infectious progeny is threefold higher in SVPi-infected cells (Fig. 1). These observations are highly reproducible and the data presented represent average values from the results of four one-step growth cycles. The results are consistent with the report that reovirus infectivity is enhanced after limited chymotrypsin digestion in the presence of specific monovalent cations (11). The SVPi lack certain virion nucleic acid (60% of the adenine-rich RNA) and capsid polypeptide ((TV, 20% of the length of ~2) components (9,101. In addition, it appears that further significant digestion and release of SVPi components may not occur after infection since the SVPi closely resemble uncoated virions isolated from infected cells (12,131. DNA synthesis was compared in SVPi- and reovirus-infected cells to determine whether the absence of release of certain virion components during the early stages of SVPi infection
260
SHORT
Id
COMMUNICATIONS
1
O---Q SVPi REOVIRUS
0
3
6
9
12
I5
IS
21
24
HOURS FIG. 1. Virus replication cycles in reovirus and SVPi- (50 PFU/cell) infected mouse L-929 cells (5 x lo5 cells/ml). The cells were cultivated in minimal essential medium with 5% heat-inactivated fetal calf serum. SVPi were prepared as described by Shatkin and La Fiandra (IO), and were not detectably contaminated with intact reovirus as determined by density gradient centrifugation and analysis of polypeptide composition by polyacrylamide gel electrophoresis. After adsorption for 2 hr at 4” the cells were washed with medium. The cells were resuspended in prewarmed medium, incubated at 37”, and sampled (2 ml) at selected times. Total PFU per ml of culture are presented.
might result in a delay of the inhibition of cellular DNA synthesis. The time at which a detectable inhibition of DNA synthesis was first observed was significantly delayed in SVPi-infected cells (Fig. 2A). Also, the degree of inhibition was reduced from 90 to 40% of control levels at 12 hr after infection. Cells were infected with 500 PFU/cell of SVPi or reovirus (Fig. 2B). Although high multiplicities of SVPi induced both an earlier and more pronounced inhibition of DNA synthesis than low multiplicities, the overall efficiency of inhibition was significantly less than that seen in cells infected with high multiplicities of complete reovirus. The reduced efficiency of DNA inhibition after SVPi infection could have been due to the lack of introduction and intracellular release of a virion component which is missing in SVPi. Subasinghe and
Loh (14) have suggested the involvement of outer capsid structures in the development of cytotoxic effects following reovirus infection under conditions of restricted virus replication. It was possible that removal or modification of capsid components might result in different cytotoxic characteristics in SVPi-infected cells compared to cells infected with intact reovirus. As a measure of the comparative rate of development of nonspecific cytotoxic effects the ability of cells to take up neutral red was determined at selected times after reovirus and SVPi infection. At 12 hr after infection the number of cells able to take up neutral red (neutral red-positive) was reduced 20-50% in the SVPi-infected culture. In contrast, the number of neutral red-positive cells was reduced 50-90% in the reovirus-infected culture (Fig. 3A). Thus, even though the efficiency of virus replication is increased, the rate of cell degeneration is significantly decreased in SVPi-infected cells. The observed differences in the number of neutral red-positive A
z 0 I
I
I
I
I
I
I B
c-4 H
15
Control Virus SVPi
IO i
*O+y-if-& 5
OI 0
I 2
I 4
I 6 TIME
I 8
I IO
I I2
(hrs)
2. Incorporation of [3H]thymidine into acidinsoluble material in cells infected with (A) low multiplicities (30 PFU/cell) and (B) high multiplicities (500 PFU/cell) of reovirus or SVPi. Samples of lo6 cells were pulse-labeled for 30 min as previously described (6). FIG.
SHORT
COMMUNICATIONS
p+==-~~ 5 - Control Id-
w I
0
A
Virus SVPi
1
I
I
2
4
6 TIME
I
I
I
8
IO
12
(hrs)
FIG. 3. Viable cell determination (A) and incorporation of [“Hlthymidine per lo’ viable cells (B). Cells were infected with reovirus or SVPi (50 PFU/ cell). Viable cells were scored as those which were positively stained with neutral red. Cells were stained with 0.005% neutral red for 30 min (A). Cells were pulse-labeled for 30 min (6) and stained for viability (B).
cells seem to account for the apparent dissimilarity in the rates of DNA synthesis in reovirus and SVPi-infected cells. When DNA synthesis was measured in reovirus and SVPi-infected cells and the results expressed in terms of [3Hlthymidine incorporation per 100 viable (neutral red-positive) cells, no difference was noted in the rate or degree of inhibition of DNA synthesis (Fig. 3B). These results suggest that nonspecific cytotoxic effects occurring after infection by intact reovirus may be responsible for a portion of the inhibition of DNA synthesis occurring during the early stages of the virus replication cycle at high multiplicities of infection. The release of capsid components or their breakdown products after infection with complete reovirus (12, 13) may account, in part, for early cytotoxic effects that are significantly reduced in cells infected with SVPi which do not contain and would not release such potentially cytotoxic components. This is con-
261
sistent with the suggestion that capsid components may be responsible for significant cytotoxicity (14). While the results presented here do not differentiate between nonspecific cytotoxic effects and specific events which may result in the inhibition of the initiation of DNA synthesis, the use of SVPi reduces early cytotoxicity and suggests that the inhibition of DNA synthesis is related to virus replication events occurring 6-8 hr after infection. This may represent the time at which a specific infection-related product(s) is synthesized or accumulates to sufficient concentration resulting in the inhibition of DNA synthesis. The use of infectious subviral particles may provide an attractive tool for further elucidation of the possible participation of a virion component or a virus replication event in the inhibition of cell DNA function after virus replication. ACKNOWLEDGMENTS These investigations were supported by The Damon Runyon Memorial Fund for Cancer Research, Grant No. DRG-1144, and the American Cancer Society, Grant No. VC-108. REFERENCES P. J., and TAMM, I., Biochim. Bio1. GOMATOS, phys. Acta 72, 651-653 (1963). 2. KUDO, H., and GRAHAM, A. F., J. Bacterial. 90, 936-945 (1965). 3. Cox, D. C. and SHAW, J. E.,J. Viral. 13.760-761 (1974). 4. HAND, R., and TAMM, I., J. Mol. Biol. 82, 174185 (1974). 5. ENSMINGER, W. D., and TAMM, I., Virology 39, 935-938 (1969). 6. SHAW, J. E., and Cox, D. C., J. Viral. 12, 704710 (1973). 7. HAND, R., and TAMM, I., J. Viral. 11, 223-231 (1973). 8. LAI, M. T., WERENNE, J. J., and JOKLIK, W. K., Virology 54, 237-244 (1973). 9. JOKLIK, W. K., Virology 49, 700-715 (1972). 10. SHATKIN, A. J., and LA FIANDRA, A. J., J. Virol. 10, 698-706 (1972). Il. BORSA, J., SARGENT, M. D., COPPS, T. P., LONG, D. G., and CHAPMAN, J. D., J. Virol. 11,10171019 (1973). 12. CHANC, C. T., and ZWEERINK, H. J., Virology 46, 544-555 (1971). 13. SILVERSTEIN, S. C., ASTELL, C., LEVIN, D. H., SCHONBERG, M., and Acs, G., Virology 47, 797-806 (1972). 14. SUBASINGHE, H. A., and LOH, P. C., Arch. Ges. Virusforsch. 39, 172-189 (1972).
74, 259-261
Infectious
(1976)
Reovirus Subviral Cytopathology, DONALD
Department
of Botany
C. COX’ and
Particles: Virus Replication, and DNA Synthesis’ C. WORTH
AND
Microbiology,
University
Accepted
July
Cellular
CLINKSCALES”
of Oklahoma,
Norman,
Oklahoma
73069
14,1976
The formation of virions in cells infected with reovirus infectious subviral particles (SVPi) appears to begin earlier, proceeds at a greater rate, and results in a threefold higher yield of progeny virus than in infections with complete virions. However, SVPi infection results in significantly reduced nonspecific cytotoxic effects. When corrections are made for cytotoxic effects, SVPi inhibit cell DNA synthesis at the same rate and to the same degree as intact reovirus.
Reovirus infection results in an inhibition of cell DNA replication (1+2). This inhibition appears to be a consequence of the prevention of the initiation of DNA synthesis (3,4) and is not related to modifications in precursor pools, DNA polymerase activity, or template integrity (5,6). An investigation of DNA synthesis in cells treated with specific subviral particles which are missing certain virion subunits could identify the viral components or replicative functions which mediate the inhibition of cell DNA synthesis. Recent studies (7,8) have shown that two noninfectious reovirus subviral particles, top component and cores, do not cause such inhibition. Reovirus subviral particles produced by limited chymotrypsin digestion lack certain capsid polypeptides but retain infectivity (9, 10). The experiments described in this report compare virus replication characteristics, modification of cell DNA synthesis, and the development of nonspecific cytotoxicity in cells treated I Portions of this work were presented at the meeting of the American Society for Microbiology, 1974, Chicago, Illinois. * Author to whom requests for reprints should be addressed. 3 Present address: Microbiology Department, University of Massachusetts Medical School, Worcester, Mass. 259 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
with complete reovirus and infectious subviral particles (SVPi). SVPi adsorb to cells as efficiently as intact reovirus (unpublished). It was necessary to determine whether this interaction was reflected in an equally efficient virus replication cycle. Significant levels of infectious virus are detectable earlier, the rate of progeny production appears to be greater, and the final yield of infectious progeny is threefold higher in SVPi-infected cells (Fig. 1). These observations are highly reproducible and the data presented represent average values from the results of four one-step growth cycles. The results are consistent with the report that reovirus infectivity is enhanced after limited chymotrypsin digestion in the presence of specific monovalent cations (11). The SVPi lack certain virion nucleic acid (60% of the adenine-rich RNA) and capsid polypeptide ((TV, 20% of the length of ~2) components (9,101. In addition, it appears that further significant digestion and release of SVPi components may not occur after infection since the SVPi closely resemble uncoated virions isolated from infected cells (12,131. DNA synthesis was compared in SVPi- and reovirus-infected cells to determine whether the absence of release of certain virion components during the early stages of SVPi infection
260
SHORT
Id
COMMUNICATIONS
1
O---Q SVPi REOVIRUS
0
3
6
9
12
I5
IS
21
24
HOURS FIG. 1. Virus replication cycles in reovirus and SVPi- (50 PFU/cell) infected mouse L-929 cells (5 x lo5 cells/ml). The cells were cultivated in minimal essential medium with 5% heat-inactivated fetal calf serum. SVPi were prepared as described by Shatkin and La Fiandra (IO), and were not detectably contaminated with intact reovirus as determined by density gradient centrifugation and analysis of polypeptide composition by polyacrylamide gel electrophoresis. After adsorption for 2 hr at 4” the cells were washed with medium. The cells were resuspended in prewarmed medium, incubated at 37”, and sampled (2 ml) at selected times. Total PFU per ml of culture are presented.
might result in a delay of the inhibition of cellular DNA synthesis. The time at which a detectable inhibition of DNA synthesis was first observed was significantly delayed in SVPi-infected cells (Fig. 2A). Also, the degree of inhibition was reduced from 90 to 40% of control levels at 12 hr after infection. Cells were infected with 500 PFU/cell of SVPi or reovirus (Fig. 2B). Although high multiplicities of SVPi induced both an earlier and more pronounced inhibition of DNA synthesis than low multiplicities, the overall efficiency of inhibition was significantly less than that seen in cells infected with high multiplicities of complete reovirus. The reduced efficiency of DNA inhibition after SVPi infection could have been due to the lack of introduction and intracellular release of a virion component which is missing in SVPi. Subasinghe and
Loh (14) have suggested the involvement of outer capsid structures in the development of cytotoxic effects following reovirus infection under conditions of restricted virus replication. It was possible that removal or modification of capsid components might result in different cytotoxic characteristics in SVPi-infected cells compared to cells infected with intact reovirus. As a measure of the comparative rate of development of nonspecific cytotoxic effects the ability of cells to take up neutral red was determined at selected times after reovirus and SVPi infection. At 12 hr after infection the number of cells able to take up neutral red (neutral red-positive) was reduced 20-50% in the SVPi-infected culture. In contrast, the number of neutral red-positive cells was reduced 50-90% in the reovirus-infected culture (Fig. 3A). Thus, even though the efficiency of virus replication is increased, the rate of cell degeneration is significantly decreased in SVPi-infected cells. The observed differences in the number of neutral red-positive A
z 0 I
I
I
I
I
I
I B
c-4 H
15
Control Virus SVPi
IO i
*O+y-if-& 5
OI 0
I 2
I 4
I 6 TIME
I 8
I IO
I I2
(hrs)
2. Incorporation of [3H]thymidine into acidinsoluble material in cells infected with (A) low multiplicities (30 PFU/cell) and (B) high multiplicities (500 PFU/cell) of reovirus or SVPi. Samples of lo6 cells were pulse-labeled for 30 min as previously described (6). FIG.
SHORT
COMMUNICATIONS
p+==-~~ 5 - Control Id-
w I
0
A
Virus SVPi
1
I
I
2
4
6 TIME
I
I
I
8
IO
12
(hrs)
FIG. 3. Viable cell determination (A) and incorporation of [“Hlthymidine per lo’ viable cells (B). Cells were infected with reovirus or SVPi (50 PFU/ cell). Viable cells were scored as those which were positively stained with neutral red. Cells were stained with 0.005% neutral red for 30 min (A). Cells were pulse-labeled for 30 min (6) and stained for viability (B).
cells seem to account for the apparent dissimilarity in the rates of DNA synthesis in reovirus and SVPi-infected cells. When DNA synthesis was measured in reovirus and SVPi-infected cells and the results expressed in terms of [3Hlthymidine incorporation per 100 viable (neutral red-positive) cells, no difference was noted in the rate or degree of inhibition of DNA synthesis (Fig. 3B). These results suggest that nonspecific cytotoxic effects occurring after infection by intact reovirus may be responsible for a portion of the inhibition of DNA synthesis occurring during the early stages of the virus replication cycle at high multiplicities of infection. The release of capsid components or their breakdown products after infection with complete reovirus (12, 13) may account, in part, for early cytotoxic effects that are significantly reduced in cells infected with SVPi which do not contain and would not release such potentially cytotoxic components. This is con-
261
sistent with the suggestion that capsid components may be responsible for significant cytotoxicity (14). While the results presented here do not differentiate between nonspecific cytotoxic effects and specific events which may result in the inhibition of the initiation of DNA synthesis, the use of SVPi reduces early cytotoxicity and suggests that the inhibition of DNA synthesis is related to virus replication events occurring 6-8 hr after infection. This may represent the time at which a specific infection-related product(s) is synthesized or accumulates to sufficient concentration resulting in the inhibition of DNA synthesis. The use of infectious subviral particles may provide an attractive tool for further elucidation of the possible participation of a virion component or a virus replication event in the inhibition of cell DNA function after virus replication. ACKNOWLEDGMENTS These investigations were supported by The Damon Runyon Memorial Fund for Cancer Research, Grant No. DRG-1144, and the American Cancer Society, Grant No. VC-108. REFERENCES P. J., and TAMM, I., Biochim. Bio1. GOMATOS, phys. Acta 72, 651-653 (1963). 2. KUDO, H., and GRAHAM, A. F., J. Bacterial. 90, 936-945 (1965). 3. Cox, D. C. and SHAW, J. E.,J. Viral. 13.760-761 (1974). 4. HAND, R., and TAMM, I., J. Mol. Biol. 82, 174185 (1974). 5. ENSMINGER, W. D., and TAMM, I., Virology 39, 935-938 (1969). 6. SHAW, J. E., and Cox, D. C., J. Viral. 12, 704710 (1973). 7. HAND, R., and TAMM, I., J. Viral. 11, 223-231 (1973). 8. LAI, M. T., WERENNE, J. J., and JOKLIK, W. K., Virology 54, 237-244 (1973). 9. JOKLIK, W. K., Virology 49, 700-715 (1972). 10. SHATKIN, A. J., and LA FIANDRA, A. J., J. Virol. 10, 698-706 (1972). Il. BORSA, J., SARGENT, M. D., COPPS, T. P., LONG, D. G., and CHAPMAN, J. D., J. Virol. 11,10171019 (1973). 12. CHANC, C. T., and ZWEERINK, H. J., Virology 46, 544-555 (1971). 13. SILVERSTEIN, S. C., ASTELL, C., LEVIN, D. H., SCHONBERG, M., and Acs, G., Virology 47, 797-806 (1972). 14. SUBASINGHE, H. A., and LOH, P. C., Arch. Ges. Virusforsch. 39, 172-189 (1972).