VIROLOGY
84, 199-202
(1978)
Cyclic Synthesis
of Human Cytomegalovirus-Induced Infected Cells PHALGUNI
GUPTA’
AND
Proteins
in
FRED RAPP’
Department of Microbiology, and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Accepted August la,1977 Synthesis of most human embryo lung cellular proteins is more sensitive to inhibition by a hypertonic condition than is synthesis of cytomegalovirus (CMOinduced proteins. Results using the selective suppression of host cell protein synthesis by the hypertonic condition indicate that virus-induced proteins are synthesized in a cyclic manner.
Human cytomegalovirus has been tonic condition than is synthesis of the virus-induced proteins. We have also shown to contain 20-30 polypeptides (I3). Very little is known about the nature found that following infection with CMV, and synthesis of the CMV polypeptides virus-induced proteins are synthesized in under in vitro and in uiuo conditions. The a cyclic manner. Mycoplasma-free human cytomegalovianalysis of CMV-induced protein synthesis in productively infected cells has been rus, strain AD169 (American Type Culcomplicated by the fact that, unlike the ture Collection) was propagated in HEL situation in herpes simplex virus (HSV)- fibroblast cells grown in Dulbecco modified infected cells (4), host protein synthesis is Eagle’s medium supplemented with 10% not suppressed during the course of CMV fetal calf serum (FCS) by the procedure described elsewhere ( 7). infection (Gupta and Rapp, unpublished In order to establish the optimum hyperobservations), and virus proteins constitute only a minor portion of the total tonic condition which would permit virusproteins synthesized in the infected cells. induced protein synthesis and minimal Several workers have shown that in polio- host protein synthesis, we compared the virus-infected (5) and simian virus 40-in- proteins synthesized in uninfected and fected (6) cells, hypertonic medium selec- CMV-infected cells late in infection. CMVtively suppresses the synthesis of most specific protein synthesis would be excellular polypeptides relative to the syn- pected to reach maximum levels midway (60 hr p.i.) or late (80-100 hr p.i.) in thesis of virus capsid proteins. In order to examine the selective sup- infection. Thus, labeling late after infecpression of host protein synthesis of CMV- tion would allow maximum detection of infected cells, we have analyzed the pat- salt-resistant CMV protein synthesis comtern of protein synthesis that occurs in pared to uninfected controls. Figure 1 illustrates that an increase in CMV-infected human embryo lung (HEL) cells during exposure to hypertonic me- the concentration of NaCl in the culture dium. The results indicate that synthesis medium resulted in a decrease in the overof the majority of host cellular proteins is all rate of l?Slmethionine incorporation more sensitive to inhibition by a hyper- into proteins by CMV- and mock-infected HEL cells. However, P5Slmethionine inI Present address: Bovine Leukemia Research, corporation by CMV-infected cells was New Bolton Center, University of Pennsylvania, consistently more resistant to hypertonic Kennet Square, Pennsylvania 19348. conditions than was incorporation by * Author to whom reprint requests should be mock-infected cells. The labeling in inaddressed. 199 0042-6822/78/0841-0199$02.00/O Copyright 0 1978 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
200
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very little incorporation by uninfected cells. Since CMV-infected cells were exposed to hypertonic treatment for only 2 hr, virus yield should not be affected particularly since CMV is slow growing and virus yield would not change significantly in 2 hr in productive infection. To characterize the proteins synthesized in CMV-infected cells in the hypertonic condition, proteins were labeled in uninfected and CMV-infected cells in 180 mM NaCl-hypertonic medium, as described in the legend to Fig. 1, and analyzed by SDS-polyacrylamide-gel electrophoresis. Figure 2 shows the presence of 18 major Externally added N&l CONCENTRATION tmMI CMV structural proteins and several nonFIG. 1. Effect of hypertonicity on the incorporastructural proteins. In contrast, very little tion of 13Y?l]methionine by CMV-infected and uninprotein was synthesized in uninfected cells fected cells. Confluent HEL cells were infected with under this condition. CMV at an input multiplicity of 1.5. Following To study the kinetics of CMV-induced virus adsorption for 2 hr at 37”, Dulbecco modified protein synthesis, infected cells were laEagle’s medium supplemented with 5% FCS was beled with l?S]methionine for 2 hr in 180 added to the cultures. At 60 hr following infection, mM NaCl-hypertonic medium at various medium was removed from both CMV- and mocktimes after infection as described in the infected cells and replaced by Joklik modified minilegend to Fig. 1. Figure 3a shows that mal essential medium supplemented with 5% FCS, nonessential amino acids, and a double complement P5S]methionine incorporation by CMV-inof vitamins (5). The medium was made hypertonic fected cells followed a cyclic pattern which by the addition of appropriate amounts of 2 M NaCl. was not observed in mock-infected cells. After 15 min of exposure to hypertonic medium at This cyclic pattern of protein synthesis 37”, [Wlmethionine (specific activity, 320 Ci/mmol; was observed in cells infected with both Amersham/Searle, Arlington Heights, Ill.) was high (1.5 PFU/cell) and low (0.2 PFU/cell) added to a final concentration of 5 &i/ml and the multiplicities of infection (m.0.i.). An cells were labeled for 2 hr at 37”. Following incorpom.o.i. greater than 1.5 could not be used ration, labeling medium was removed and cells because it causes rapid cytopathology rewere solubilized by boiling in a buffer containing sulting in very low virus yield and cell 0.0625 M Tris, 2% SDS, and 5% mercaptoethanol for 2 min, and an aliquot of each sample was measured death. At an m.o.i. of 1.5, all cells showed for trichloroacetic acid-precipitable radioactivity. cytopathic effects in 48 hr indicating that all cells were infected. In order to determine whether the obfected cells was 14 times higher than in uninfected cells in the presence of a 180 served cyclic pattern of CMV-induced protein synthesis is an artifact of hypertonic mM final concentration of externally added NaCl. Because the incorporation of treatment during P5Slmethionine incorr5S]methionine in both uninfected and poration, protein synthesis was measured in CMV-infected cells in normal (isotonic) CMV-infected cells is severely inhibited labeling medium. Figure 3b shows that, at an externally added NaCl concentration higher than 180 mM, we used that concen- although the incorporation due to cellular tration (hereafter designated 180 mM protein synthesis was very high, a cyclic NaCl-hypertonic medium) to study the nature of protein synthesis was observed synthesis of CMV-induced proteins in in- in infected cells, particularly in late stages fected cells. Under this hypertonic condi- of infection. Data presented in this communication tion, although both uninfected and infected cells are viable (data not shown), suggest that the hypertonic conditions produced by the addition of NaCl suppressed CMV-infected cells incorporate significant amounts of P5S]methionine, in contrast to the labeling of most of the host polypep-
SHORT
201
COMMUNICATIONS
and therefore would be expected to stimulate host protein synthesis. .As shown in Fig. 2, the proteins synthesized after infection include CMV structural and nonstructural proteins. Since very few of these proteins were detected in uninfected controls, we conclude that the proteins synthesized in CMV-infected cells in the presence of hypertonic medium are primarily
FIG. 2. Polyacrylamide-gel electrophoresis analysis of proteins synthesized in uninfected and CMVinfected cells in the presence of hypertonic medium. Uninfected and CMV-infected HEL cells were labeled with [35S]methionine in the presence of 180 m&f NaCl-hypertonic medium, as described in the legend to Fig. 1. Samples were solubilized as described in the legend to Fig. 1 and analyzed by electrophoresis in a 5-15% gradient polyacrylamide gel in the presence of SDS (3). Following electrophoresis, the gel was dried and an autoradiogram was prepared using Kodak No-Screen film. (a) Uninfected cell protein; (b) CMV-infected cell protein; (c) S5S-labeled purified CMV polypeptides. Numbers (I-18) indicate major CMV structural proteins.
tides relative to the labeling of CMV polypeptides. Under the conditions used in these experiments, 10 to 15 times more radioactivity was incorporated in CMV-infected cells than in uninfected cells. It has been reported that CMV infection stimulates host DNA and RNA synthesis (8, 9)
0
40 80 120 Hours After Infection FIG. 3. (a) CMV-induced protein synthesis in infected cells exposed to NaCl-hypertonic medium. At various times after infection, mock-infected or CMV-infected cells were exposed to 180 mM NaClhypertonic medium for 15 min followed by labeling with [Wlmethionine for 2 hr as described in the legend to the Fig. 1. The cells were solubilized and trichloroacetic acid-precipitable radioactivity was determined as described in the legend to Fig. 1. Protein synthesis in cells infected with CMV at an input multiplicity of 1.5 (A) or 0.2 (0); (0) and (Cl) represent corresponding HEL controls. (b) Rate of protein synthesis in CMV-infected cells in the absence of a hypertonic condition. At various times after infection, mock-infected (0) or CMV-infected (A) cells were exposed to Joklik modified synthetic medium for 15 min and labeled with 13Wmethionine for 2 hr (final concentration of 5 pCi/ml). Following incorporation, proteins were solubilized and trichloroacetic acid-precipitable radioactivity was determined as described in the legend to Fig. 1.
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virus specific. We do not know which of these proteinq are virus-genome coded and which are coded by the host genome. It has been well documented that the new proteins synthesized after HSV type 2 infection also constitute 24 virus structural proteins as well as 27 nonstructural proteins (41, for which the genomic origin is still unknown. The selective suppression of host cell protein synthesis by the hypertonic condition offers a unique opportunity to study the synthesis of CMV-induced proteins after infection. Our results indicate that, following infection with CMV, proteins are synthesized in a cyclic manner. Although the cyclic pattern is reproducible, the time required for the cycle to repeat varies between 25 and 40 hr. One explanation is that CMV infection synchronized the cells. Although synchronization of HEL cells following CMV infection has not been reported, there are reports indicating that CMV infection of HEL cells results in a cyclic synthesis of both virus and cellular DNA (10). It would be interesting to compare protein synthesis after infection with other slow-growing herpesviruses, such as Epstein-Barr virus or varicella-zoster virus.
ACKNOWLEDGMENTS We thank Dr. Mary K. Howett for helpful discussions. This work was supported by Contract No. NO1 CP53516 within the Virus Cancer Program of the National Cancer Institute, NIH, PHS, and by Grant No. 1 P30 CA18450, awarded by the National Cancer Institute, DHEW. REFERENCES 1. FIALA, M., HONESS, R. W., HEINER, D. C., HEINE, J. W., JR., MURNANE, J., WALLACE, R., and GUZE, L. B., J. Viral. 19, 243-254 (1976). 2. SAROV I., and ABADY, J., Virology 66, 464-473 (1975). 3. GUPTA, P., ST. JEOR, S., and RAPP, F., J. Gen. Viral. 34, 447-454 (1977). 4. POWELL, K. L., and COURTNEY, R. J., Virology 66, 217-228 (1975). 5. Nuss, D. L., OPPERMANN, H., and KOCH, G., Proc. Nat. Acad. Sci. USA 72, 1258-1262 (1975). 6. ENGLAND, J. M., HOWETT, M. K., and TAN, K. B., J. Viral. 16, 1101-1107 (1975). 7. ST. JEOR, S. C., and RAPP, F., J. Viral. 11, 986990 (1973). 8. ST. JEOR, S. C., ALBRECHT, T. B., FUNK, F. D., and RAPP, F., J. Viral. 13,353-362 (1974). 9. TANAKA, S., FURUKAWA, T., and PLOTKIN, S. A., J. Viral. 15, 297-304 (1975). 10. ST. JEOR, S. C., and HUTT, R., J. Gen. Viral., in press.
84, 199-202
(1978)
Cyclic Synthesis
of Human Cytomegalovirus-Induced Infected Cells PHALGUNI
GUPTA’
AND
Proteins
in
FRED RAPP’
Department of Microbiology, and Specialized Cancer Research Center, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Accepted August la,1977 Synthesis of most human embryo lung cellular proteins is more sensitive to inhibition by a hypertonic condition than is synthesis of cytomegalovirus (CMOinduced proteins. Results using the selective suppression of host cell protein synthesis by the hypertonic condition indicate that virus-induced proteins are synthesized in a cyclic manner.
Human cytomegalovirus has been tonic condition than is synthesis of the virus-induced proteins. We have also shown to contain 20-30 polypeptides (I3). Very little is known about the nature found that following infection with CMV, and synthesis of the CMV polypeptides virus-induced proteins are synthesized in under in vitro and in uiuo conditions. The a cyclic manner. Mycoplasma-free human cytomegalovianalysis of CMV-induced protein synthesis in productively infected cells has been rus, strain AD169 (American Type Culcomplicated by the fact that, unlike the ture Collection) was propagated in HEL situation in herpes simplex virus (HSV)- fibroblast cells grown in Dulbecco modified infected cells (4), host protein synthesis is Eagle’s medium supplemented with 10% not suppressed during the course of CMV fetal calf serum (FCS) by the procedure described elsewhere ( 7). infection (Gupta and Rapp, unpublished In order to establish the optimum hyperobservations), and virus proteins constitute only a minor portion of the total tonic condition which would permit virusproteins synthesized in the infected cells. induced protein synthesis and minimal Several workers have shown that in polio- host protein synthesis, we compared the virus-infected (5) and simian virus 40-in- proteins synthesized in uninfected and fected (6) cells, hypertonic medium selec- CMV-infected cells late in infection. CMVtively suppresses the synthesis of most specific protein synthesis would be excellular polypeptides relative to the syn- pected to reach maximum levels midway (60 hr p.i.) or late (80-100 hr p.i.) in thesis of virus capsid proteins. In order to examine the selective sup- infection. Thus, labeling late after infecpression of host protein synthesis of CMV- tion would allow maximum detection of infected cells, we have analyzed the pat- salt-resistant CMV protein synthesis comtern of protein synthesis that occurs in pared to uninfected controls. Figure 1 illustrates that an increase in CMV-infected human embryo lung (HEL) cells during exposure to hypertonic me- the concentration of NaCl in the culture dium. The results indicate that synthesis medium resulted in a decrease in the overof the majority of host cellular proteins is all rate of l?Slmethionine incorporation more sensitive to inhibition by a hyper- into proteins by CMV- and mock-infected HEL cells. However, P5Slmethionine inI Present address: Bovine Leukemia Research, corporation by CMV-infected cells was New Bolton Center, University of Pennsylvania, consistently more resistant to hypertonic Kennet Square, Pennsylvania 19348. conditions than was incorporation by * Author to whom reprint requests should be mock-infected cells. The labeling in inaddressed. 199 0042-6822/78/0841-0199$02.00/O Copyright 0 1978 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
200
SHORT
COMMUNICATIONS
very little incorporation by uninfected cells. Since CMV-infected cells were exposed to hypertonic treatment for only 2 hr, virus yield should not be affected particularly since CMV is slow growing and virus yield would not change significantly in 2 hr in productive infection. To characterize the proteins synthesized in CMV-infected cells in the hypertonic condition, proteins were labeled in uninfected and CMV-infected cells in 180 mM NaCl-hypertonic medium, as described in the legend to Fig. 1, and analyzed by SDS-polyacrylamide-gel electrophoresis. Figure 2 shows the presence of 18 major Externally added N&l CONCENTRATION tmMI CMV structural proteins and several nonFIG. 1. Effect of hypertonicity on the incorporastructural proteins. In contrast, very little tion of 13Y?l]methionine by CMV-infected and uninprotein was synthesized in uninfected cells fected cells. Confluent HEL cells were infected with under this condition. CMV at an input multiplicity of 1.5. Following To study the kinetics of CMV-induced virus adsorption for 2 hr at 37”, Dulbecco modified protein synthesis, infected cells were laEagle’s medium supplemented with 5% FCS was beled with l?S]methionine for 2 hr in 180 added to the cultures. At 60 hr following infection, mM NaCl-hypertonic medium at various medium was removed from both CMV- and mocktimes after infection as described in the infected cells and replaced by Joklik modified minilegend to Fig. 1. Figure 3a shows that mal essential medium supplemented with 5% FCS, nonessential amino acids, and a double complement P5S]methionine incorporation by CMV-inof vitamins (5). The medium was made hypertonic fected cells followed a cyclic pattern which by the addition of appropriate amounts of 2 M NaCl. was not observed in mock-infected cells. After 15 min of exposure to hypertonic medium at This cyclic pattern of protein synthesis 37”, [Wlmethionine (specific activity, 320 Ci/mmol; was observed in cells infected with both Amersham/Searle, Arlington Heights, Ill.) was high (1.5 PFU/cell) and low (0.2 PFU/cell) added to a final concentration of 5 &i/ml and the multiplicities of infection (m.0.i.). An cells were labeled for 2 hr at 37”. Following incorpom.o.i. greater than 1.5 could not be used ration, labeling medium was removed and cells because it causes rapid cytopathology rewere solubilized by boiling in a buffer containing sulting in very low virus yield and cell 0.0625 M Tris, 2% SDS, and 5% mercaptoethanol for 2 min, and an aliquot of each sample was measured death. At an m.o.i. of 1.5, all cells showed for trichloroacetic acid-precipitable radioactivity. cytopathic effects in 48 hr indicating that all cells were infected. In order to determine whether the obfected cells was 14 times higher than in uninfected cells in the presence of a 180 served cyclic pattern of CMV-induced protein synthesis is an artifact of hypertonic mM final concentration of externally added NaCl. Because the incorporation of treatment during P5Slmethionine incorr5S]methionine in both uninfected and poration, protein synthesis was measured in CMV-infected cells in normal (isotonic) CMV-infected cells is severely inhibited labeling medium. Figure 3b shows that, at an externally added NaCl concentration higher than 180 mM, we used that concen- although the incorporation due to cellular tration (hereafter designated 180 mM protein synthesis was very high, a cyclic NaCl-hypertonic medium) to study the nature of protein synthesis was observed synthesis of CMV-induced proteins in in- in infected cells, particularly in late stages fected cells. Under this hypertonic condi- of infection. Data presented in this communication tion, although both uninfected and infected cells are viable (data not shown), suggest that the hypertonic conditions produced by the addition of NaCl suppressed CMV-infected cells incorporate significant amounts of P5S]methionine, in contrast to the labeling of most of the host polypep-
SHORT
201
COMMUNICATIONS
and therefore would be expected to stimulate host protein synthesis. .As shown in Fig. 2, the proteins synthesized after infection include CMV structural and nonstructural proteins. Since very few of these proteins were detected in uninfected controls, we conclude that the proteins synthesized in CMV-infected cells in the presence of hypertonic medium are primarily
FIG. 2. Polyacrylamide-gel electrophoresis analysis of proteins synthesized in uninfected and CMVinfected cells in the presence of hypertonic medium. Uninfected and CMV-infected HEL cells were labeled with [35S]methionine in the presence of 180 m&f NaCl-hypertonic medium, as described in the legend to Fig. 1. Samples were solubilized as described in the legend to Fig. 1 and analyzed by electrophoresis in a 5-15% gradient polyacrylamide gel in the presence of SDS (3). Following electrophoresis, the gel was dried and an autoradiogram was prepared using Kodak No-Screen film. (a) Uninfected cell protein; (b) CMV-infected cell protein; (c) S5S-labeled purified CMV polypeptides. Numbers (I-18) indicate major CMV structural proteins.
tides relative to the labeling of CMV polypeptides. Under the conditions used in these experiments, 10 to 15 times more radioactivity was incorporated in CMV-infected cells than in uninfected cells. It has been reported that CMV infection stimulates host DNA and RNA synthesis (8, 9)
0
40 80 120 Hours After Infection FIG. 3. (a) CMV-induced protein synthesis in infected cells exposed to NaCl-hypertonic medium. At various times after infection, mock-infected or CMV-infected cells were exposed to 180 mM NaClhypertonic medium for 15 min followed by labeling with [Wlmethionine for 2 hr as described in the legend to the Fig. 1. The cells were solubilized and trichloroacetic acid-precipitable radioactivity was determined as described in the legend to Fig. 1. Protein synthesis in cells infected with CMV at an input multiplicity of 1.5 (A) or 0.2 (0); (0) and (Cl) represent corresponding HEL controls. (b) Rate of protein synthesis in CMV-infected cells in the absence of a hypertonic condition. At various times after infection, mock-infected (0) or CMV-infected (A) cells were exposed to Joklik modified synthetic medium for 15 min and labeled with 13Wmethionine for 2 hr (final concentration of 5 pCi/ml). Following incorporation, proteins were solubilized and trichloroacetic acid-precipitable radioactivity was determined as described in the legend to Fig. 1.
202
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virus specific. We do not know which of these proteinq are virus-genome coded and which are coded by the host genome. It has been well documented that the new proteins synthesized after HSV type 2 infection also constitute 24 virus structural proteins as well as 27 nonstructural proteins (41, for which the genomic origin is still unknown. The selective suppression of host cell protein synthesis by the hypertonic condition offers a unique opportunity to study the synthesis of CMV-induced proteins after infection. Our results indicate that, following infection with CMV, proteins are synthesized in a cyclic manner. Although the cyclic pattern is reproducible, the time required for the cycle to repeat varies between 25 and 40 hr. One explanation is that CMV infection synchronized the cells. Although synchronization of HEL cells following CMV infection has not been reported, there are reports indicating that CMV infection of HEL cells results in a cyclic synthesis of both virus and cellular DNA (10). It would be interesting to compare protein synthesis after infection with other slow-growing herpesviruses, such as Epstein-Barr virus or varicella-zoster virus.
ACKNOWLEDGMENTS We thank Dr. Mary K. Howett for helpful discussions. This work was supported by Contract No. NO1 CP53516 within the Virus Cancer Program of the National Cancer Institute, NIH, PHS, and by Grant No. 1 P30 CA18450, awarded by the National Cancer Institute, DHEW. REFERENCES 1. FIALA, M., HONESS, R. W., HEINER, D. C., HEINE, J. W., JR., MURNANE, J., WALLACE, R., and GUZE, L. B., J. Viral. 19, 243-254 (1976). 2. SAROV I., and ABADY, J., Virology 66, 464-473 (1975). 3. GUPTA, P., ST. JEOR, S., and RAPP, F., J. Gen. Viral. 34, 447-454 (1977). 4. POWELL, K. L., and COURTNEY, R. J., Virology 66, 217-228 (1975). 5. Nuss, D. L., OPPERMANN, H., and KOCH, G., Proc. Nat. Acad. Sci. USA 72, 1258-1262 (1975). 6. ENGLAND, J. M., HOWETT, M. K., and TAN, K. B., J. Viral. 16, 1101-1107 (1975). 7. ST. JEOR, S. C., and RAPP, F., J. Viral. 11, 986990 (1973). 8. ST. JEOR, S. C., ALBRECHT, T. B., FUNK, F. D., and RAPP, F., J. Viral. 13,353-362 (1974). 9. TANAKA, S., FURUKAWA, T., and PLOTKIN, S. A., J. Viral. 15, 297-304 (1975). 10. ST. JEOR, S. C., and HUTT, R., J. Gen. Viral., in press.
