VIROLOGY
70, 238-240 (1976)
Effect of Infection
with Enveloped
G. HAMMER, Znstitut
Viruses
R. T. SCHWARZ,
fiir Virologie,
AND
on Nucleotide C. SCHOLTISSEK
Justus Liebig-Universitiit,
Accepted November
Metabolism
Giessen, Germany
24, 1975
After infection of primary chick embryo or BHK cells with Semliki Forest, Newcastle disease, pseudorabies, or fowl plague virus t,he maximal velocities of the uptake of uridine, cytidine, and guanosine, but not that of adenosine were markedly enhanced, while the apparent K, values were not affected. The increase in the V,,,, values is paralleled by a corresponding enhancement of the nucleoside kinase activities measured in the cytosol of infected cells. Only after infection with Semliki Forest virus also was a significant decrease of the absolute pool sizes of the nucleoside triphosphates found. Therefore, in Semliki Forest virus-infected BHK cells the stimulation of the uptake of uridine is not only caused by changes of the membrane function but also by the release of a negative feedback regulation controlled by the UTP pool.
Enveloped animal viruses mature by budding at the cell periphery. There is no sudden death of the cells by a burst as found, e.g., for nonenveloped viruses. In some cases there is even no shut-off of the host metabolism. Therefore, the very complex reactions leading to the cytopathic effect are still obscure. In this paper, we have studied changes in the uptake of nucleosides into primary chick embryo or BHK cells and of the nucleotide pools after infection with Newcastle disease (NDV), fowl plague (FPV), Semliki Forest (SFV), and pseudorabies (PRV) viruses in order to get some information on altered membrane functions and of the energy state of these cells after infection. The following virus strains were investigated: Newcastle disease virus, strains “Italien” (NDV-I) or “Beaudette” (NDVB); fowl plaque virus (FPV), strain “Rostock”; Semliki Forest virus (SFV), strain virus, “Osterrieth”; and pseudorabies strain “Dekking” (PRV). Primary chick embryo cells prepared from 11-day-old embryos were used 24 hr after seeding. These cells or BHK-21 cells were infected with the various virus strains at a multiplicity of infection between 10 and 50 PFU/cell. If not otherwise stated, cells were kept after infection at 37”
in Earle’s medium without serum. The in uivo labeling experiments were performed by incorporation of [53Hluridine or L3Hladenosine (uniformly labeled) at various specific activities as described recently (7). Nucleoside kinase activities were determined by the conversion of labeled nucleosides into the corresponding triphosphates using a cytosol fraction (6). The absolute pools of uracil nucleotides were determined in the neutralized perchloric acid extracts (total volume, 1 ml) prepared from 10’ cells according to Keppler et al. (4). The CTP- and CDPcholine pools were measured by the isotope dilution technique according to Domschke et al. (3) starting with 10” cells. In Table 1 data on the apparent K, and relative I’,,, values on the uptake of uridine and adenosine into chick embryo cells infected with various enveloped viruses are summarized. These data were obtained from Lineweaver-Burk plots of cells pulse-labeled (7) at the maximal rate of virus multiplication (for SFV- and FPVinfected cultures at 4 hr; for NDV-infected cultures at 6 hr, and for PRV-infected cultures at 12 hr after infection). Also, values on the relative kinase activities in the cytosole of cells harvested at the same times were included in Table 1. It can be seen 238
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
239
COMMUNICATIONS TABLE
1
APPARENT K,,, AND RELATIVE V,,, VALUES ON THE UPTAKE OF URIDINE AND ADENOSINE INTO CHICK EMBRYO CELLS AND URIDINE AND ADENOSINE KINASE ACTIVITIES AFTER INFECTION WITH VARIOUS ENVELOPED VIRUSES~ -___Virus Uridine Adenosine Kinase (N/No)
NDV-I NDV-B FPV SFV PRV
1.0 1.0 1.2 09 1.0
3.4 1.45 2.9 1.7 2.7
2.5 2.2 1.5 -
1.0 1.1 0.8 1.1 1.0
Vm,, (N/ No)
Kinase (N/No)
1.0 1.0 1.0 1.0 1.0
0.99 0.98 0.97 --V,,, values (N/ cultures.
‘I The K,,t and V,,, values were calculated from Lineweaver-Burk plots(7). The relative No) are the values obtained from infected cultures over those obtained from mock-infected
that the maximal velocity of the uptake of uridine is mostly stimulated in NDV-I-infected cells, while the stimulation after SFV-infection is less pronounced. There is also a striking difference between the highly pathogenic NDV strain “Italien” and the less pathogenic strain “Baudette.” In BHK cells, the V,,, value was enhanced after SFV infection by a factor of 4.5, while there was no stimulation after infection with NDV-I (data not shown). Thus, this effect seems to depend also on the host cells under investigation. There was no significant change of the apparent K, values between infected and mock-infected cells (error of the method is 20.2 x lo-” M). These results are interpreted to mean that the number of sites (V,,,) may be increased after infection, but not the affinity of uridine to the transport system (KJ. Similar observations have been made concerning the uptake of cytidine and guanosine. As shown in Table 1, adenosine uptake, however, is not affected by infection. Since it is assumed that the nucleoside kinases play a specific role in the transport of nucleosides (5, 7) also, kinase activities were measured in infected and mock-infected cultures. The results included in Table 1 further support the concept that the number of the available sites for uridine transport may be increased by infection. The relative distribution of the radioactivity among the various nucleotides WTP, UDP, UMP, UDP-sugars, ATP, ADP, and AMP) after labeling with
t3Hluridine or i3Hladenosine, respectively, did not change after infection with FPV or NDV. However, after infection with SFV radioactivity accumulated in the monoand diphosphate fractions, which is in agreement with the observation by Cassels and Burke (1) that the energy charge of the host cells is affected by the infection with SFV. Therefore, we have studied in more detail the effect of virus infection on the absolute pools of various nucleotides. As can be seen in Table 2, the absolute pools of UTP and UDP-glucose decrease in mock-infected cells in dependence of the time after changing the medium. There is, however, no additional decrease caused by infection with NDV or FPV. An additional marked decrease in only SFV-infected cells has been observed in chick embryo cells as well as in BHK cells. Corresponding data were obtained concerning the CTP- and CDP-choline pools (4 hr after SFV-infection decrease by 85 or 70%, respectively, but no decrease in NDV-infected cells). The same observations were made concerning the guanosine nucleotide pools (not shown here). Cunningham and Pardee (2) have shown that serum has a marked effect on the uptake of uridine into 3T3 cells. The same is true for chick embryo cells as well as for BHK cells (decrease of uptake by a factor of 2 or 3 at 4 or 6 hr after change of medium containing 10% calf serum to serum-free medium, respectively). It has to be stressed, therefore, that the marked increase in uptake of various nucleosides by
240
SHORT
COMMUNICATIONS TABLE
ABSOLUTE
Experiment and cell type
Infection with
1 CEC
Mock
FPV
Mock SFV
3 BHK
Nucleotide
Mock SFV NDV
U All values represent
2
UTP UDP-Gl UTP UDP-Gl UTP UDP-GI
3
12.8 4.8 10.8 4.3 12.0 4.2
UTP UDP-Gl UTP UDP-Gl
23.5 8.1 17.6 9.4
UTP UDP-Gl UTP UDP-Gl UTP UDP-GI
12.6 5.8 11.9 5.3
nanomoles/:!
AND
Hours after infection 1
NDV
2 CEC
2
POOL SIZES OF UTP AND LJDP-GLUCOSE (LJDP-Gl) IN CHICK EMBRYO (CEC) BHK-CELLS AT DIFFERENT TIMES AFTER INFECTION WITH VARIOUS VIRUSES
17.6 5.8 8.1 4.6
14.2 5.2 6.4 3.0
4
6
9.0 4.0 8.3 3.2 8.1 3.5
7.0 2.9 7.8 3.0 9.0 2.9
7
0
7.0 2.7 7.7 2.6 7.1 2.6
12.4 5.1 4.2 2.8
10.2 4.7 3.1 2.4
7.5 4.4
7.3 3.7
x 10’ cells.
infection does not occur in medium containing 10% calf serum and that the alterations in the plasma membrane functions can be unraveled only by omission of the serum by induction of a suboptimal condition for the transport of uridine, cytidine and guanosine. Adenosine always is transported optimally under the conditions employed. Therefore, its uptake is not influenced by infection in serum-free medium. It is known that the UTP pool regulates the uptake of uridine as well as &dine by negative feedback (8). Thus, two different mechanisms may be responsible for the enhanced uptake of certain nucleosides after virus infection: Either there is an alteration of the membrane structure (most clearly shown in NDV-infected chick embryo cells, where no change of the UTP pool is induced by infection) or the enhancement is caused by release of a negative feedback via a decrease of the corresponding nucleoside triphosphate pools (most clearly shown in SFV-infected chick embryo cells). In SFV-infected BHK cells, both mechanisms seem to act concurrently.
ACKNOWLEDGMENTS We thank Dr. Rott for his continuous interest and helpful discussion. The skilful technical assistance of Mrs. I. Weibel is gratefully acknowledged. This work was supported by the Sonderforschungsbereich 47 (Virologiel and the Fonds der Chemischen Industrie. This work is presented as a partial fulltillment of the requirements for the doctoral thesis (1975) of G. Hammer (D 26; Fachbereich Biologie, Justus Liebig-Universitat, Giessen). REFERENCES 1. CASSELS, A. C., and BURKE, D. C., J. Gen. Viral. 18, 135-141 (1973). 2. CUNNINGHAM, D. D., and PARDEE, A. B., Proc. Nut. Acad. Sci. USA 64, 1049-1056 (19691. 3. DOMSCHKE, W., KEPPLER, D., BISCHOFF, E., and DECKER, K., Hoppe-Seyler’s 2. Physiol. Chem. 352, 275-279 (1971). 4. KEPPLER, D., RUDIGIER, J., and DECKER, K., Anal. Biochem. 38, 105-114 (1970). 5. RAU, J., and SCHOLTISSEK, C., 2. Naturforsch. 25b, 292-299 (1970). 6. SCHOLTISSEK, C., Biochim. Biophys. Actu 155, 14-23 (1968). 7. SCHOLTISSEK, C., Biochim. Biophys. Actu 158, 435-447 (1968). 8. SCHOLTISSEK, C., Biochim. Biophys. Acta 277, 459-465 (1972).
70, 238-240 (1976)
Effect of Infection
with Enveloped
G. HAMMER, Znstitut
Viruses
R. T. SCHWARZ,
fiir Virologie,
AND
on Nucleotide C. SCHOLTISSEK
Justus Liebig-Universitiit,
Accepted November
Metabolism
Giessen, Germany
24, 1975
After infection of primary chick embryo or BHK cells with Semliki Forest, Newcastle disease, pseudorabies, or fowl plague virus t,he maximal velocities of the uptake of uridine, cytidine, and guanosine, but not that of adenosine were markedly enhanced, while the apparent K, values were not affected. The increase in the V,,,, values is paralleled by a corresponding enhancement of the nucleoside kinase activities measured in the cytosol of infected cells. Only after infection with Semliki Forest virus also was a significant decrease of the absolute pool sizes of the nucleoside triphosphates found. Therefore, in Semliki Forest virus-infected BHK cells the stimulation of the uptake of uridine is not only caused by changes of the membrane function but also by the release of a negative feedback regulation controlled by the UTP pool.
Enveloped animal viruses mature by budding at the cell periphery. There is no sudden death of the cells by a burst as found, e.g., for nonenveloped viruses. In some cases there is even no shut-off of the host metabolism. Therefore, the very complex reactions leading to the cytopathic effect are still obscure. In this paper, we have studied changes in the uptake of nucleosides into primary chick embryo or BHK cells and of the nucleotide pools after infection with Newcastle disease (NDV), fowl plague (FPV), Semliki Forest (SFV), and pseudorabies (PRV) viruses in order to get some information on altered membrane functions and of the energy state of these cells after infection. The following virus strains were investigated: Newcastle disease virus, strains “Italien” (NDV-I) or “Beaudette” (NDVB); fowl plaque virus (FPV), strain “Rostock”; Semliki Forest virus (SFV), strain virus, “Osterrieth”; and pseudorabies strain “Dekking” (PRV). Primary chick embryo cells prepared from 11-day-old embryos were used 24 hr after seeding. These cells or BHK-21 cells were infected with the various virus strains at a multiplicity of infection between 10 and 50 PFU/cell. If not otherwise stated, cells were kept after infection at 37”
in Earle’s medium without serum. The in uivo labeling experiments were performed by incorporation of [53Hluridine or L3Hladenosine (uniformly labeled) at various specific activities as described recently (7). Nucleoside kinase activities were determined by the conversion of labeled nucleosides into the corresponding triphosphates using a cytosol fraction (6). The absolute pools of uracil nucleotides were determined in the neutralized perchloric acid extracts (total volume, 1 ml) prepared from 10’ cells according to Keppler et al. (4). The CTP- and CDPcholine pools were measured by the isotope dilution technique according to Domschke et al. (3) starting with 10” cells. In Table 1 data on the apparent K, and relative I’,,, values on the uptake of uridine and adenosine into chick embryo cells infected with various enveloped viruses are summarized. These data were obtained from Lineweaver-Burk plots of cells pulse-labeled (7) at the maximal rate of virus multiplication (for SFV- and FPVinfected cultures at 4 hr; for NDV-infected cultures at 6 hr, and for PRV-infected cultures at 12 hr after infection). Also, values on the relative kinase activities in the cytosole of cells harvested at the same times were included in Table 1. It can be seen 238
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
239
COMMUNICATIONS TABLE
1
APPARENT K,,, AND RELATIVE V,,, VALUES ON THE UPTAKE OF URIDINE AND ADENOSINE INTO CHICK EMBRYO CELLS AND URIDINE AND ADENOSINE KINASE ACTIVITIES AFTER INFECTION WITH VARIOUS ENVELOPED VIRUSES~ -___Virus Uridine Adenosine Kinase (N/No)
NDV-I NDV-B FPV SFV PRV
1.0 1.0 1.2 09 1.0
3.4 1.45 2.9 1.7 2.7
2.5 2.2 1.5 -
1.0 1.1 0.8 1.1 1.0
Vm,, (N/ No)
Kinase (N/No)
1.0 1.0 1.0 1.0 1.0
0.99 0.98 0.97 --V,,, values (N/ cultures.
‘I The K,,t and V,,, values were calculated from Lineweaver-Burk plots(7). The relative No) are the values obtained from infected cultures over those obtained from mock-infected
that the maximal velocity of the uptake of uridine is mostly stimulated in NDV-I-infected cells, while the stimulation after SFV-infection is less pronounced. There is also a striking difference between the highly pathogenic NDV strain “Italien” and the less pathogenic strain “Baudette.” In BHK cells, the V,,, value was enhanced after SFV infection by a factor of 4.5, while there was no stimulation after infection with NDV-I (data not shown). Thus, this effect seems to depend also on the host cells under investigation. There was no significant change of the apparent K, values between infected and mock-infected cells (error of the method is 20.2 x lo-” M). These results are interpreted to mean that the number of sites (V,,,) may be increased after infection, but not the affinity of uridine to the transport system (KJ. Similar observations have been made concerning the uptake of cytidine and guanosine. As shown in Table 1, adenosine uptake, however, is not affected by infection. Since it is assumed that the nucleoside kinases play a specific role in the transport of nucleosides (5, 7) also, kinase activities were measured in infected and mock-infected cultures. The results included in Table 1 further support the concept that the number of the available sites for uridine transport may be increased by infection. The relative distribution of the radioactivity among the various nucleotides WTP, UDP, UMP, UDP-sugars, ATP, ADP, and AMP) after labeling with
t3Hluridine or i3Hladenosine, respectively, did not change after infection with FPV or NDV. However, after infection with SFV radioactivity accumulated in the monoand diphosphate fractions, which is in agreement with the observation by Cassels and Burke (1) that the energy charge of the host cells is affected by the infection with SFV. Therefore, we have studied in more detail the effect of virus infection on the absolute pools of various nucleotides. As can be seen in Table 2, the absolute pools of UTP and UDP-glucose decrease in mock-infected cells in dependence of the time after changing the medium. There is, however, no additional decrease caused by infection with NDV or FPV. An additional marked decrease in only SFV-infected cells has been observed in chick embryo cells as well as in BHK cells. Corresponding data were obtained concerning the CTP- and CDP-choline pools (4 hr after SFV-infection decrease by 85 or 70%, respectively, but no decrease in NDV-infected cells). The same observations were made concerning the guanosine nucleotide pools (not shown here). Cunningham and Pardee (2) have shown that serum has a marked effect on the uptake of uridine into 3T3 cells. The same is true for chick embryo cells as well as for BHK cells (decrease of uptake by a factor of 2 or 3 at 4 or 6 hr after change of medium containing 10% calf serum to serum-free medium, respectively). It has to be stressed, therefore, that the marked increase in uptake of various nucleosides by
240
SHORT
COMMUNICATIONS TABLE
ABSOLUTE
Experiment and cell type
Infection with
1 CEC
Mock
FPV
Mock SFV
3 BHK
Nucleotide
Mock SFV NDV
U All values represent
2
UTP UDP-Gl UTP UDP-Gl UTP UDP-GI
3
12.8 4.8 10.8 4.3 12.0 4.2
UTP UDP-Gl UTP UDP-Gl
23.5 8.1 17.6 9.4
UTP UDP-Gl UTP UDP-Gl UTP UDP-GI
12.6 5.8 11.9 5.3
nanomoles/:!
AND
Hours after infection 1
NDV
2 CEC
2
POOL SIZES OF UTP AND LJDP-GLUCOSE (LJDP-Gl) IN CHICK EMBRYO (CEC) BHK-CELLS AT DIFFERENT TIMES AFTER INFECTION WITH VARIOUS VIRUSES
17.6 5.8 8.1 4.6
14.2 5.2 6.4 3.0
4
6
9.0 4.0 8.3 3.2 8.1 3.5
7.0 2.9 7.8 3.0 9.0 2.9
7
0
7.0 2.7 7.7 2.6 7.1 2.6
12.4 5.1 4.2 2.8
10.2 4.7 3.1 2.4
7.5 4.4
7.3 3.7
x 10’ cells.
infection does not occur in medium containing 10% calf serum and that the alterations in the plasma membrane functions can be unraveled only by omission of the serum by induction of a suboptimal condition for the transport of uridine, cytidine and guanosine. Adenosine always is transported optimally under the conditions employed. Therefore, its uptake is not influenced by infection in serum-free medium. It is known that the UTP pool regulates the uptake of uridine as well as &dine by negative feedback (8). Thus, two different mechanisms may be responsible for the enhanced uptake of certain nucleosides after virus infection: Either there is an alteration of the membrane structure (most clearly shown in NDV-infected chick embryo cells, where no change of the UTP pool is induced by infection) or the enhancement is caused by release of a negative feedback via a decrease of the corresponding nucleoside triphosphate pools (most clearly shown in SFV-infected chick embryo cells). In SFV-infected BHK cells, both mechanisms seem to act concurrently.
ACKNOWLEDGMENTS We thank Dr. Rott for his continuous interest and helpful discussion. The skilful technical assistance of Mrs. I. Weibel is gratefully acknowledged. This work was supported by the Sonderforschungsbereich 47 (Virologiel and the Fonds der Chemischen Industrie. This work is presented as a partial fulltillment of the requirements for the doctoral thesis (1975) of G. Hammer (D 26; Fachbereich Biologie, Justus Liebig-Universitat, Giessen). REFERENCES 1. CASSELS, A. C., and BURKE, D. C., J. Gen. Viral. 18, 135-141 (1973). 2. CUNNINGHAM, D. D., and PARDEE, A. B., Proc. Nut. Acad. Sci. USA 64, 1049-1056 (19691. 3. DOMSCHKE, W., KEPPLER, D., BISCHOFF, E., and DECKER, K., Hoppe-Seyler’s 2. Physiol. Chem. 352, 275-279 (1971). 4. KEPPLER, D., RUDIGIER, J., and DECKER, K., Anal. Biochem. 38, 105-114 (1970). 5. RAU, J., and SCHOLTISSEK, C., 2. Naturforsch. 25b, 292-299 (1970). 6. SCHOLTISSEK, C., Biochim. Biophys. Actu 155, 14-23 (1968). 7. SCHOLTISSEK, C., Biochim. Biophys. Actu 158, 435-447 (1968). 8. SCHOLTISSEK, C., Biochim. Biophys. Acta 277, 459-465 (1972).
