VIROLOGY
66, 322-326 (1975)
SHORT Studies
COMMUNICATIONS
on the Synthesis Complexes
of Viral RNA-Polymerase-Template
in BHK 21 Cells Infected
with
Semliki
Forest Virus GERD WENGLER Znstitut fir
Virologie,
GISELA WENGLER
AND
Justus Liebig-Universitiit, Accepted Februury
Giessen, Germany
17,1975
Two main types of virus-specific RNA sedimenting at about 42 and 26 S, respectively, on sucrose density gradients are synthesized in animal cells infected with Semliki Forest virus (SFV). Two and one-quarter hours after infection of BHK 21 cells with SFV only about 10% of the total amount of virus-specific RNA that accumulates during the 8-hr growth cycle of the virus have been synthesized. If at this time an inhibitor of protein synthesis, cycloheximide, is added to infected cells, both of these RNA species are synthesized during the viral growth cycle with the same time course and in amounts similar to those in untreated cultures. This result suggests that the RNA polymerase-template complexes present at 2.25 hr post infection (p.i.) are stable and synthesize all virus-specific 42 and 26 S RNA accumulating during the viral growth cycle. These polymerase-template complexes are synthesized between 45 min and 2.25 hr p.i. During multiplication of SFV in BHK 21 cells the amount of radioactivity incorporated from radioactively labeled amino acids into protein during a series of labeling intervals decreases, and the ratio of synthesis of virus-specific proteins to cellular proteins increases. Both of these effects are not expressed to a significant extent at 2.25 hr post infection (p.i.). Sodium dodecyl sulfate-polyacrylamide-gel electrophoretic (SDS-PAGE) analysis of the polypeptides synthesized in infected cultures shortly after removal of cycloheximide, which had been present from 2.25-6 hr p.i. shows that cycloheximide does not block the development of these changes in cellular protein synthesis. Some implications of these findings concerning the synthesis of virus-specific RNA and the interference of virus multiplication with cellular protein synthesis are discussed.
A virus-induced RNA-dependent RNApolymerase activity is detectable in extracts of cells infected with the group A arbovirus Semliki Forest virus (SFV) or Sindbis virus and some properties of the enzyme have been described (1-5). In the present communication evidence is presented that the polymerase-template complexes in BHK 21 cells infected with SFV are fully active during prolonged treatment of the cultures with an inhibitor of protein synthesis, cycloheximide. The time of synthesis of polymerase-template complexes in infected cells has been measured and the effect of temporary cycloheximide treat-
ment on the virus-induced changes of cellular protein synthesis has been determined. The techniques used in the present experiments have been described (6, 7). SFV multiplies in BHK 21 cells treated with actinomycin D (1 pg/ml). Since this concentration of actinomycin D blocks synthesis of cellular RNA almost completely, the virus-specific RNA synthesized in these cells can be labeled with radioactive nucleosides rather specifically and the synthesis of virus-specific RNA can be followed. In the experiment presented in Fig. 1 the influence of the presence of cycloheximide (50 &ml) on the time course of
322 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
323
SHORT COMMUNICATIONS
incorporation of [9H]uridine into virusspecific RNA in SFV-infected BHK 21 cells has been determined. The dose of cycloheximide used inhibits cellular and viral protein synthesis by more than 90% during the whole interval of drug treatment (data not shown). It can be seen from the data presented in Fig. 1 that the effect of cycloheximide on viral RNA synthesis is dependent on the time of addition of the drug. If cycloheximide is added at 2.25 hr p.i., a time at which only about 10% of the total virus-specific RNA which accumulates in infected cultures during the viral growth cycle has been made, [3H @idine is incorporated into virus-specific RNA during the rest of the viral growth cycle as in infected cultures not treated with the drug. If cycloheximide is added at 45 min p.i. incorporation of [3H]uridine into RNA is almost totally inhibited and a partial inhibition of synthesis of virus-specific RNA is seen if cycloheximide is added at 1 hr 30 min p.i. That the incorporation of [3H]uridine into acid-precipitabie material reflects the synthesis of virus-specific RNA can be concluded from the experiment presented in Fig. 2. The RNA molecules synthesized in the presence or absence of cycloheximide in SFV-infected actinomycin D-treated BHK 21 cells were isolated and fractionated by sucrose density gradient centrifugation. The two main species of virusspecific RNA which are synthesized in untreated cells and which sediment at about 42 and 26 S, respectively, accumulate in cycloheximide-treated cells in amounts similar to those in the untreated cells. The 42 S RNA is detectable in the optical density analyses of the sucrose density gradients in a region well separated from the ribosoma128 S RNA. That the 42 S RNA molecules made in cycloheximidetreated and in untreated cultures are not complementary to each other but of the same polarity can be concluded from the fact that both are about equally infectious (data not shown). From the results presented in Fig. 1 it can be then concluded that at 2.25 hr p.i. polymerase-template complexes sufficient
LO c
.-
12
-
1
I
3
1
hours
/
5
. I
6
/
7
I
a
p I
FIG. 1. Influence of cycloheximide on the incorpo-
ration of [3H]uridine into virus-specific RNA in BHK 21 cells infected with SFV. Freshly confluent monolayer cultures containing about 6 x 10’ cells each were infected with SFV (about 100 PFU/cell). All media and buffers used contained actinomycin D (1 fig/ml). After the 30-min period used for infection (time 0), all cultures received fresh growth medium containing actinomycin D (1 rg/ml), [SHjuridine (5 &i/ml) and unlabeled uridine (30 &Z) and were incubated further at 37”. The cultures were then divided into four aliquots: The first series of cultures was left untreated; to the growth medium of the second, third and fourth series cycloheximide was added to 50 rg/ml at 45 min, 1 hr 30 min and 2 hr 15 min p.i., respectively. Cultures were taken from all four aliquots at 45-min intervals, and the amount of radioactivity incorporated into acid-precipitable material was determined. The radioactivity incorporated in the different cultures is indicated as follows: LO, Untreated cultures; m----m, cycloheximide added at 45 min p.i.; A-A, cycloheximide added at 1 hr 30 min p.i.; x-x cycloheximide added at 2 hr 15 min pi.
for the synthesis of a normal complement of virus-specific RNA are already present in the infected cells and that these complexes are synthesized between 45 min and 2.25 hr p.i. Two major changes take place in protein synthesis in BHK 21 cell cultures infected with SFV (B-11): The amount of radioactivity incorporated into total protein during labeling intervals with radioactive amino acids decreases, and increasing amounts of virus-specific peptides are synthesized, until at about 6 hr p.i. the majority of newly synthesized proteins are virus-specific
324
SHORT COMMUNICATIONS
FIG. 2. Sucrose density gradient analysis of virusspecific RNA synthesized in SFV-infected BHK 21 cells in the absence or presence of cycloheximide. Freshly confluent monolayer cultures were infected with SFV in the presence of actinomycin D as described in the legend to Fig. 1. After the 30-min period used for this treatment (time 0), all cultures were incubated with fresh growth medium containing actinomycin D (1 &ml) and unlabeled uridine (30 PM). One series of cultures was left untreated; to the growth medium of a second series, cycloheximide was added at 2 hr 15 min p.i. At the same time [*H ]uridine was added to the growth medium of all cultures to a final concentration of 5 &i/ml. All cultures were incubated at 37Ofor a further 4 hr 30 min. At 6 hr 45 min p.i. cells were harvested from all cultures, and the RNA was extracted immediately with phenol at 60’. Similar aliquots of the RNA isolated from both series of cultures were fractionated by centrifugation on 10-30s (w/w) sucrose density gradients in buffer containing 10 mM K acetate, 50 mM KCl, 0.1 mM Na EDTA pH 5.2. Centrifugation was done for 6 hr at 40,000 rpm at 2” in an SW 41 Spinco rotor. The optical density distribution in the gradients was determined in a flow-through cuvette, and radioactivity was measured in an aliquot of each fraction. (A) Fractionation of RNA from untreated cultures; (B), fractionation of RNA from cultures treated with cycloheximide. -, Optical density; O--O, [aH juridine radioactivity.
(Figs. 3A, B, and C). At 2.25 hr p.i. these effects are not significantly expressed (Figs. 3A and B), but, since at this time enough polymerase-template complexes have accumulated to sustain RNA syn-
thesis in the presence of cycloheximide, an experiment can be made to measure whether or not a block of protein synthesis by cycloheximide from 2.25-6 hr p.i. inhibits the immediate expression of the above mentioned virus-induced changes in protein synthesis shortly after removal of the cycloheximide. The results of such an experiment are presented in Fig. 3. It can be seen that both effects of virus infection on protein synthesis have developed in cycloheximide-treated infected cells. No significant difference can be detected between the protein synthesized in mock-infected cells before or after a similar treatment with cycloheximide (see Fig. 3A for the fractionation of the proteins labeled before cycloheximide treatment) (data not shown). The results presented above suggest that the RNA polymerase-template complexes present in infected cells at 2.25 hr p.i., a time at which only about 10% of the total virus-specific RNA accumulating in infected cells has been made, do not turn over and are responsible for the synthesis of the virus-specific 42 and 26 S RNA during the rest of the viral growth cycle. In cells treated at 2.25 hr p.i. with cycloheximide, virus-specific 42 and 26 S RNA is synthesized during the following hours as in untreated cultures. No evidence is therefore found in this experiment for the existence of two different polymerase-template complexes producing either 42 or 26 S RNA. The results are compatible with the models of RNA synthesis suggested by Simmons and Strauss (12) or by Martin and Burke (13) in which 26 S RNA and 42 S RNA are both synthesized from an RNA template complementary to the 42 S RNA which contains more than one site for initiation and/or termination of RNA synthesis and with the assumption that the different sites for RNA synthesis initiation and/or termination can be recognized in each polymerase-template complex. Treatment of infected cells with cycloheximide between 2.25 and 6 hr p.i. does not inhibit the expression of the two major virus-induced changes in cellular protein synthesis: The reduction of incorporation
SHORT
325
COMMUNICATIONS
Fraction
number
FIG. 3. Influence of cycloheximide on the development of virus-induced alterations of cellular protein synthesis. Freshly confluent monolayer cultures were infected with SFV as described in the legend to Fig. 1 or mock infected. After the 30-min period used for this treatment (time 0), all cultures were further incubated with fresh growth medium containing actinomycin D (1 fig/ml) at 37”. Infected and mock-infected cultures were each divided into two aliquots: Aliquot one was left untreated; to the growth medium of aliquot two cycloheximide was added at 2 hr 15 min p.i. At 6 hr p.i. the growth medium was removed from all cultures, and all cultures were washed four times with growth medium at 37” (this is sufficient to remove the cycloheximide from the cultures treated with this drug). Fresh growth medium was then added, and the cultures were further incubated at 37”. At different times after infection cultures were taken from the four aliquots and the newly synthesized proteins were labeled with [aH]leucine by addition to the medium of [8H]leucine to 10 rCi/ml. After 20-min incubation of the cultures at 37”, the cells were harvested and stored at -80” as pellets. Cells were prepared for SDS-PAGE and analyzed on 7.5% SDS-polyacrylamide gels as described earlier (7). Direction of migration of the proteins during electrophoresis is from left to right. The virus specific proteins are C, core protein (14); E, the two envelope glycoproteins E, and E, which are not resolved under the electrophoretic conditions used in this experiment (15); NSP 68, a nonstructural virus specific protein that is a precursor of the viral glycoprotein E, (10). Direction of migration of the proteins during electrophoresis is from left to right.(A),Mock-infected cells labeled from 1 hr 55 min to 2 hr 15 min p.i.; (B), virus-infected cells labeled from 1 hr 55 min to 2 hr 15 min p.i.; (C), virus-infected cultures not treated with cycloheximide, labeled from 6 hr 15 min to 6 hr 35 min pi.; (D),virus-infected cultures incubated in the presence of cycloheximide from 2 hr 15 min to 6 hr p.i. and labeled in the absence of cycloheximide (see above) from 6 hr 15 min to 6 hr 35 min p.i. O---O, [3H]leucine radioactivity.
from labeled amino acids and the increase in the relative amounts of virus-specific as compared to cellular proteins synthesized in infected cultures. Continuous synthesis of a protein which would either enzymatically or nonenzymatically modify the cellular apparatus for translation or the cellular mRNA is therefore not necessary for the development of these changes in cellular protein synthesis. The production of such a protein during the first 2.25 hr after infection which, for example, could then enzymatitally function during the following hours
of radioactivity into protein
cannot be excluded. The results are on the other hand easily compatible with the assumption that synthesis and/or accumulation of virus-specific RNA are responsible for the virus-induced alterations in cellular protein synthesis. Experiments aimed at elucidating the processes involved in the virus-induced changes of cellular protein synthesis are currently underway in our laboratory. ACKNOLWEDGMENTS We express our gratitude to Dr. R. Rott for interest and support. We thank L. Rohrschneider for critical
326
SHORT
COMMUNICATIONS
reading of the manuscript. This study was supported by the Sonderforschungsbereich 47 (Virologie).
Virology 61, 120-128 (1974). 8. STRAUSSI. H., JR., BURGE,B. W., and DARNELL,I. E., Virology 37, 367-376.
REFERENCES I. A., J. Viral. 1, 1. MARTIN, E. M., and SONNABEND,
97-m
(1967).
2. MARTIN, E. M., Virology 39, 107-117 (1969). 3. Q-HI, A. A., and TRENT, D. W., J. Viral. 9, 565-573 (1972). 4. FRIEDMAN, R. M., LEVIN, I. G., GRIMLEY,P. M., and BEREZESKY,I. K., J. Viral. 10, 504-515 (1972). 5. MICHEL, M. R., and GOMATOS,P. I., J. Viral. 11, 900-914 (1973). 6. WENGLER, G., and WENGLER, G., Virolo&‘y 59, 21-35 (1974). 7. WENGLER,G., BEATO,M., and HACKEMACK,B.-A.,
C. I., MARTIN, E. M., and COOPER,P. M., J. Gen. Virol. 6, 319-323 (1970). 10. SIMONS, K., KERXNEN, S., and KXXRIXINEN,L., FEBS Lett. 29,87-91(1973). 11. MORSER, R. I., and BURKED. C., J. Gem Viral. 22, 9. BIJRRU,
395-409 (1974).
12. SIMMONS, D. T., and STRAUSS,I. H., J. Mol. Bid. 71, 615-631 (1972).
13. MARTIN,B. A. B., and BURKE,D. C., J. Gen. Viral. 24, 45-66 (1974).
14. ACHESON, N. H., and TAMM, I., Virology 41, 321-329 (1970). 15. GAROW, H., SIMONS, K., and RFNKONEN, O., Virology 61, 493-504 (1974).
66, 322-326 (1975)
SHORT Studies
COMMUNICATIONS
on the Synthesis Complexes
of Viral RNA-Polymerase-Template
in BHK 21 Cells Infected
with
Semliki
Forest Virus GERD WENGLER Znstitut fir
Virologie,
GISELA WENGLER
AND
Justus Liebig-Universitiit, Accepted Februury
Giessen, Germany
17,1975
Two main types of virus-specific RNA sedimenting at about 42 and 26 S, respectively, on sucrose density gradients are synthesized in animal cells infected with Semliki Forest virus (SFV). Two and one-quarter hours after infection of BHK 21 cells with SFV only about 10% of the total amount of virus-specific RNA that accumulates during the 8-hr growth cycle of the virus have been synthesized. If at this time an inhibitor of protein synthesis, cycloheximide, is added to infected cells, both of these RNA species are synthesized during the viral growth cycle with the same time course and in amounts similar to those in untreated cultures. This result suggests that the RNA polymerase-template complexes present at 2.25 hr post infection (p.i.) are stable and synthesize all virus-specific 42 and 26 S RNA accumulating during the viral growth cycle. These polymerase-template complexes are synthesized between 45 min and 2.25 hr p.i. During multiplication of SFV in BHK 21 cells the amount of radioactivity incorporated from radioactively labeled amino acids into protein during a series of labeling intervals decreases, and the ratio of synthesis of virus-specific proteins to cellular proteins increases. Both of these effects are not expressed to a significant extent at 2.25 hr post infection (p.i.). Sodium dodecyl sulfate-polyacrylamide-gel electrophoretic (SDS-PAGE) analysis of the polypeptides synthesized in infected cultures shortly after removal of cycloheximide, which had been present from 2.25-6 hr p.i. shows that cycloheximide does not block the development of these changes in cellular protein synthesis. Some implications of these findings concerning the synthesis of virus-specific RNA and the interference of virus multiplication with cellular protein synthesis are discussed.
A virus-induced RNA-dependent RNApolymerase activity is detectable in extracts of cells infected with the group A arbovirus Semliki Forest virus (SFV) or Sindbis virus and some properties of the enzyme have been described (1-5). In the present communication evidence is presented that the polymerase-template complexes in BHK 21 cells infected with SFV are fully active during prolonged treatment of the cultures with an inhibitor of protein synthesis, cycloheximide. The time of synthesis of polymerase-template complexes in infected cells has been measured and the effect of temporary cycloheximide treat-
ment on the virus-induced changes of cellular protein synthesis has been determined. The techniques used in the present experiments have been described (6, 7). SFV multiplies in BHK 21 cells treated with actinomycin D (1 pg/ml). Since this concentration of actinomycin D blocks synthesis of cellular RNA almost completely, the virus-specific RNA synthesized in these cells can be labeled with radioactive nucleosides rather specifically and the synthesis of virus-specific RNA can be followed. In the experiment presented in Fig. 1 the influence of the presence of cycloheximide (50 &ml) on the time course of
322 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
323
SHORT COMMUNICATIONS
incorporation of [9H]uridine into virusspecific RNA in SFV-infected BHK 21 cells has been determined. The dose of cycloheximide used inhibits cellular and viral protein synthesis by more than 90% during the whole interval of drug treatment (data not shown). It can be seen from the data presented in Fig. 1 that the effect of cycloheximide on viral RNA synthesis is dependent on the time of addition of the drug. If cycloheximide is added at 2.25 hr p.i., a time at which only about 10% of the total virus-specific RNA which accumulates in infected cultures during the viral growth cycle has been made, [3H @idine is incorporated into virus-specific RNA during the rest of the viral growth cycle as in infected cultures not treated with the drug. If cycloheximide is added at 45 min p.i. incorporation of [3H]uridine into RNA is almost totally inhibited and a partial inhibition of synthesis of virus-specific RNA is seen if cycloheximide is added at 1 hr 30 min p.i. That the incorporation of [3H]uridine into acid-precipitabie material reflects the synthesis of virus-specific RNA can be concluded from the experiment presented in Fig. 2. The RNA molecules synthesized in the presence or absence of cycloheximide in SFV-infected actinomycin D-treated BHK 21 cells were isolated and fractionated by sucrose density gradient centrifugation. The two main species of virusspecific RNA which are synthesized in untreated cells and which sediment at about 42 and 26 S, respectively, accumulate in cycloheximide-treated cells in amounts similar to those in the untreated cells. The 42 S RNA is detectable in the optical density analyses of the sucrose density gradients in a region well separated from the ribosoma128 S RNA. That the 42 S RNA molecules made in cycloheximidetreated and in untreated cultures are not complementary to each other but of the same polarity can be concluded from the fact that both are about equally infectious (data not shown). From the results presented in Fig. 1 it can be then concluded that at 2.25 hr p.i. polymerase-template complexes sufficient
LO c
.-
12
-
1
I
3
1
hours
/
5
. I
6
/
7
I
a
p I
FIG. 1. Influence of cycloheximide on the incorpo-
ration of [3H]uridine into virus-specific RNA in BHK 21 cells infected with SFV. Freshly confluent monolayer cultures containing about 6 x 10’ cells each were infected with SFV (about 100 PFU/cell). All media and buffers used contained actinomycin D (1 fig/ml). After the 30-min period used for infection (time 0), all cultures received fresh growth medium containing actinomycin D (1 rg/ml), [SHjuridine (5 &i/ml) and unlabeled uridine (30 &Z) and were incubated further at 37”. The cultures were then divided into four aliquots: The first series of cultures was left untreated; to the growth medium of the second, third and fourth series cycloheximide was added to 50 rg/ml at 45 min, 1 hr 30 min and 2 hr 15 min p.i., respectively. Cultures were taken from all four aliquots at 45-min intervals, and the amount of radioactivity incorporated into acid-precipitable material was determined. The radioactivity incorporated in the different cultures is indicated as follows: LO, Untreated cultures; m----m, cycloheximide added at 45 min p.i.; A-A, cycloheximide added at 1 hr 30 min p.i.; x-x cycloheximide added at 2 hr 15 min pi.
for the synthesis of a normal complement of virus-specific RNA are already present in the infected cells and that these complexes are synthesized between 45 min and 2.25 hr p.i. Two major changes take place in protein synthesis in BHK 21 cell cultures infected with SFV (B-11): The amount of radioactivity incorporated into total protein during labeling intervals with radioactive amino acids decreases, and increasing amounts of virus-specific peptides are synthesized, until at about 6 hr p.i. the majority of newly synthesized proteins are virus-specific
324
SHORT COMMUNICATIONS
FIG. 2. Sucrose density gradient analysis of virusspecific RNA synthesized in SFV-infected BHK 21 cells in the absence or presence of cycloheximide. Freshly confluent monolayer cultures were infected with SFV in the presence of actinomycin D as described in the legend to Fig. 1. After the 30-min period used for this treatment (time 0), all cultures were incubated with fresh growth medium containing actinomycin D (1 &ml) and unlabeled uridine (30 PM). One series of cultures was left untreated; to the growth medium of a second series, cycloheximide was added at 2 hr 15 min p.i. At the same time [*H ]uridine was added to the growth medium of all cultures to a final concentration of 5 &i/ml. All cultures were incubated at 37Ofor a further 4 hr 30 min. At 6 hr 45 min p.i. cells were harvested from all cultures, and the RNA was extracted immediately with phenol at 60’. Similar aliquots of the RNA isolated from both series of cultures were fractionated by centrifugation on 10-30s (w/w) sucrose density gradients in buffer containing 10 mM K acetate, 50 mM KCl, 0.1 mM Na EDTA pH 5.2. Centrifugation was done for 6 hr at 40,000 rpm at 2” in an SW 41 Spinco rotor. The optical density distribution in the gradients was determined in a flow-through cuvette, and radioactivity was measured in an aliquot of each fraction. (A) Fractionation of RNA from untreated cultures; (B), fractionation of RNA from cultures treated with cycloheximide. -, Optical density; O--O, [aH juridine radioactivity.
(Figs. 3A, B, and C). At 2.25 hr p.i. these effects are not significantly expressed (Figs. 3A and B), but, since at this time enough polymerase-template complexes have accumulated to sustain RNA syn-
thesis in the presence of cycloheximide, an experiment can be made to measure whether or not a block of protein synthesis by cycloheximide from 2.25-6 hr p.i. inhibits the immediate expression of the above mentioned virus-induced changes in protein synthesis shortly after removal of the cycloheximide. The results of such an experiment are presented in Fig. 3. It can be seen that both effects of virus infection on protein synthesis have developed in cycloheximide-treated infected cells. No significant difference can be detected between the protein synthesized in mock-infected cells before or after a similar treatment with cycloheximide (see Fig. 3A for the fractionation of the proteins labeled before cycloheximide treatment) (data not shown). The results presented above suggest that the RNA polymerase-template complexes present in infected cells at 2.25 hr p.i., a time at which only about 10% of the total virus-specific RNA accumulating in infected cells has been made, do not turn over and are responsible for the synthesis of the virus-specific 42 and 26 S RNA during the rest of the viral growth cycle. In cells treated at 2.25 hr p.i. with cycloheximide, virus-specific 42 and 26 S RNA is synthesized during the following hours as in untreated cultures. No evidence is therefore found in this experiment for the existence of two different polymerase-template complexes producing either 42 or 26 S RNA. The results are compatible with the models of RNA synthesis suggested by Simmons and Strauss (12) or by Martin and Burke (13) in which 26 S RNA and 42 S RNA are both synthesized from an RNA template complementary to the 42 S RNA which contains more than one site for initiation and/or termination of RNA synthesis and with the assumption that the different sites for RNA synthesis initiation and/or termination can be recognized in each polymerase-template complex. Treatment of infected cells with cycloheximide between 2.25 and 6 hr p.i. does not inhibit the expression of the two major virus-induced changes in cellular protein synthesis: The reduction of incorporation
SHORT
325
COMMUNICATIONS
Fraction
number
FIG. 3. Influence of cycloheximide on the development of virus-induced alterations of cellular protein synthesis. Freshly confluent monolayer cultures were infected with SFV as described in the legend to Fig. 1 or mock infected. After the 30-min period used for this treatment (time 0), all cultures were further incubated with fresh growth medium containing actinomycin D (1 fig/ml) at 37”. Infected and mock-infected cultures were each divided into two aliquots: Aliquot one was left untreated; to the growth medium of aliquot two cycloheximide was added at 2 hr 15 min p.i. At 6 hr p.i. the growth medium was removed from all cultures, and all cultures were washed four times with growth medium at 37” (this is sufficient to remove the cycloheximide from the cultures treated with this drug). Fresh growth medium was then added, and the cultures were further incubated at 37”. At different times after infection cultures were taken from the four aliquots and the newly synthesized proteins were labeled with [aH]leucine by addition to the medium of [8H]leucine to 10 rCi/ml. After 20-min incubation of the cultures at 37”, the cells were harvested and stored at -80” as pellets. Cells were prepared for SDS-PAGE and analyzed on 7.5% SDS-polyacrylamide gels as described earlier (7). Direction of migration of the proteins during electrophoresis is from left to right. The virus specific proteins are C, core protein (14); E, the two envelope glycoproteins E, and E, which are not resolved under the electrophoretic conditions used in this experiment (15); NSP 68, a nonstructural virus specific protein that is a precursor of the viral glycoprotein E, (10). Direction of migration of the proteins during electrophoresis is from left to right.(A),Mock-infected cells labeled from 1 hr 55 min to 2 hr 15 min p.i.; (B), virus-infected cells labeled from 1 hr 55 min to 2 hr 15 min p.i.; (C), virus-infected cultures not treated with cycloheximide, labeled from 6 hr 15 min to 6 hr 35 min pi.; (D),virus-infected cultures incubated in the presence of cycloheximide from 2 hr 15 min to 6 hr p.i. and labeled in the absence of cycloheximide (see above) from 6 hr 15 min to 6 hr 35 min p.i. O---O, [3H]leucine radioactivity.
from labeled amino acids and the increase in the relative amounts of virus-specific as compared to cellular proteins synthesized in infected cultures. Continuous synthesis of a protein which would either enzymatically or nonenzymatically modify the cellular apparatus for translation or the cellular mRNA is therefore not necessary for the development of these changes in cellular protein synthesis. The production of such a protein during the first 2.25 hr after infection which, for example, could then enzymatitally function during the following hours
of radioactivity into protein
cannot be excluded. The results are on the other hand easily compatible with the assumption that synthesis and/or accumulation of virus-specific RNA are responsible for the virus-induced alterations in cellular protein synthesis. Experiments aimed at elucidating the processes involved in the virus-induced changes of cellular protein synthesis are currently underway in our laboratory. ACKNOLWEDGMENTS We express our gratitude to Dr. R. Rott for interest and support. We thank L. Rohrschneider for critical
326
SHORT
COMMUNICATIONS
reading of the manuscript. This study was supported by the Sonderforschungsbereich 47 (Virologie).
Virology 61, 120-128 (1974). 8. STRAUSSI. H., JR., BURGE,B. W., and DARNELL,I. E., Virology 37, 367-376.
REFERENCES I. A., J. Viral. 1, 1. MARTIN, E. M., and SONNABEND,
97-m
(1967).
2. MARTIN, E. M., Virology 39, 107-117 (1969). 3. Q-HI, A. A., and TRENT, D. W., J. Viral. 9, 565-573 (1972). 4. FRIEDMAN, R. M., LEVIN, I. G., GRIMLEY,P. M., and BEREZESKY,I. K., J. Viral. 10, 504-515 (1972). 5. MICHEL, M. R., and GOMATOS,P. I., J. Viral. 11, 900-914 (1973). 6. WENGLER, G., and WENGLER, G., Virolo&‘y 59, 21-35 (1974). 7. WENGLER,G., BEATO,M., and HACKEMACK,B.-A.,
C. I., MARTIN, E. M., and COOPER,P. M., J. Gen. Virol. 6, 319-323 (1970). 10. SIMONS, K., KERXNEN, S., and KXXRIXINEN,L., FEBS Lett. 29,87-91(1973). 11. MORSER, R. I., and BURKED. C., J. Gem Viral. 22, 9. BIJRRU,
395-409 (1974).
12. SIMMONS, D. T., and STRAUSS,I. H., J. Mol. Bid. 71, 615-631 (1972).
13. MARTIN,B. A. B., and BURKE,D. C., J. Gen. Viral. 24, 45-66 (1974).
14. ACHESON, N. H., and TAMM, I., Virology 41, 321-329 (1970). 15. GAROW, H., SIMONS, K., and RFNKONEN, O., Virology 61, 493-504 (1974).
