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Selective failure of protein synthesis in herpesvirus-infected cells deprived of arginine
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COMMUNICATIONS
14.
JARRETT, 0. and MACPHERSON, I. Intern. J. Cancer 3, 654662 (1968). 16. FAWCETT, D. W. An Atlas of Fine Structure. Saunders, Philadelphia, Pennsylvania (1966). A. F. Cancer Res. 16. DALES, S. and HOWATSON, 21, 193-197 (1961). 17. KINDIG, D. A. and KIRSTEN, W. H. Science 155, 1543-1545 (1967). 18. CROMACK, A. S. J. Gen. Viral. 2, 195198 (1968). 19. MUSSGAY, M., RECZKO, E. and AHL, R. J. Gen. Viral. 4, 445447 (1969). CHARLES SHIPMAN, JR. GRETCHEN C. VANDER WEIDE BOOE IL MA~ Department of Oral Riology School of Dentistry and Department of Microbiology School of Medicine The University of Michigan Ann Arbor, Michigan .@lOJ Accepted
May
RO, 1969
1 Public DE42829-01).
Health
Special
Selective
Failure
of Protein
Herpesvirus-Infected
Fellow
Cells
(No.
Synthesis
F03
in
Deprived
of Arginine’ The yield of several DNA viruses (l-4), including that of herpes simplex virus (5-8), from animal cells in continuous cultivation is largely dependent on the presence of arginine in the medium. The requirement for arginine is more stringent than that for other amino acids (5) but it is not uniform; the yield of herpes simplex virus from primary cultures of human embryonic fibroblasts, chick embryo fibroblasts, or of monkey kidney cells is unaffected by arginine deprivation (6). The differences between primary and continuous cell cultures with respect to the capacity to support virus multiplication in arginine-free medium may reflect the size of the arginine pool (9-11) . Arginine starvation of cells in continuous cultivation does not 1 Aided by grants from the National Cancer Institute, U. S. Public Health Service (CA O&194), the American Cancer Society (E 314E), and the National Science Foundation (GB 8242).
prevent adsorption, penetration, uncoating of herpes simplex virus, and reproductive events occurring during the first several hours after infection. The latter include the synthesis of viral DNA (9) and the synthesis of viral proteins and their transport from cytoplasm into the nucleus as evidenced by the presence of viral antigens in both compartments of the cell (8). However, immunofluorescent granules in the cytoplasm and nucleus and viral particles are not made (8). These findings suggest that arginine deprivation causes a selective decrease in the synthesis of some viral macromolecules. One hypothesis that could explain these findings is based on the assumption that in infected cells depleted of arginine the translation of mRNA comes to a halt at codons specifying arginine. It could be expected therefore that in arginine-depleted cells the probability that a given peptide is completed would be inversely proportional to its molecular weight and to its arginine content. The hypothesis is of particular interest in view of the evidence presented by us and others that herpesvirus contains proteins ranging up to 125,000 daltons (12, 1s). To test this hypothesis we compared the size distribution of proteins made in untreated and arginine-deprived infected cells. In these experiments replicate HEp-2 cell cultures, each containing 2 X lo7 cells, were exposed for 1 hour at 37” to sufficient virus to yield a multiplicity of 50 plaque-forming units per cell. One set of cultures (control) was then overlaid with mixture 199 containing 1% dialyzed calf serum (199-I). The other set was overlaid with the same medium but lacking arginine (199-1 urg-). At 6 hours postinfection the maintenance medium was decanted, the cells were washed, and replenished as follows: Control cultures received medium 199-I lacking lysine (199-I Zys-) but supplemented with 0.2 PCi of 14C-l-lysine per ml of medium. The arginine-deprived cultures were replenished with medium 199-1 arg-Lys supplemented with 2 &i of 3H-llysine per ml of medium. The final concentration of lysine in the two sets was approximately the same. The cells were then reincubated. The time of labeling was based on the observations that host polyribosomes
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o+-
01 d...,.........r.r
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711
deprived infected cells were layered on 5.7 % acrylamide gels 20 cm long and electrophoresed for 18 hours. The gels were then sliced and radioactivity measured as previously described (16). To discriminate between the effects of arginine deprivation and the effects of partial lysine deprivation necessary to label the proteins, we repeated the experiments twice. In the second experiment we used media 199-l arg-leu- and 199-1 i&u- supplemented with 3H-l-leucine and 14C-l-leucine, respectively. In the third experiment we used 199-l arg- isoleu- and 199-l isoleu- supplemented with 3H-l-isoleu and 14C-1-isoleu, respectively. The choice of isoleucine, leucine, and lysine was predicated on the report by Tankersley (5) showing that the deprivation of these amino 1 1EUClNE
FIG. 1. Electrophoretic profiles of artificial mixtures of (i) proteins labeled with single 3H amino acids (either lysine, leucine, or isoleucine) in herpes simplex virus-infected HEp-2 cells deprived of arginine, and (ii) proteins labeled with the corresponding W amino acid in untreated infected cells. The cells were deprived of arginine immediately after the l-hour exposure to virus. The proteins were labeled between 6 and 20 hours after infection. Previous studies had demonstrated that host protein synthesis ceases at 4 hours postinfection (12, 14, 15). The procedures for preparation of the labeled cell lysate, electrophoresis, and countingof radioactivity in the gel slices were the same as previously described (12). The profile of radioactivity obtained with radioactive leucine is the same as previously described (12).
become disaggregated and proteins characteristic of uninfected cells ceaseto be made within 4 hours after infection (l&14,15). At 20 hours postinfection the cells were allowed to swell in distilled water and broken by 20 strokes of a tight Dounce homogenizer. The entire lysate was then solubilized in urea, sodium dodecyl sulfate, and p mercaptoethanol as previously described (12). Artificial mixtures of lysates of control and arginine-
FIG. 2. The ratio of 3H and 1% proteins in each gel slice computed from the profiles shown in Fig. 1 plotted as a function of the molecular weight of the protein in the gel. The molecular weights of proteins in the gel were estimated by co-electrophoresis of proteins made in infected cells with proteins of known size (12). For the gels used in this study there is a linear relationship between protein molecular weight and distance of migration plotted on a log scale (17).
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COMMUNICATIONS
acids has only a slight effect on virus yield. the number of arginine residues (i.e., given The profiles of radioactivity in the acrylathe same arginine frequency, shorter promide gels are shown in Fig. 1. Figure 2 shows teins have a higher probability of being comthe 3H/14C ratios computed from 3H and 14C pleted). (iii) Arginine deprivation may be more detrimental to virus multiplication counts of each gel slice and plotted as a function of the molecular weight of the proteins in than that of other amino acids because sevthe gels as determined by co-electrophoresis eral proteins specified by the virus are both large in size and rich in arginine. with proteins of known size (12, 17). It would be expected that if amino acid deprivation REFERENCES caused a decrease in the synthesis of all 1. ROUSE, H. C., BONIFAS, V. H., and proteins the 3H/14C ratio would remain relaSCHLDSINGER, R. W., Virology 30,357 (1963). tively constant. The data summarized in 2. ROUSE, H. C., and SCHLESINGER, R. W., ViFig. 2 show that (i) the ratios of 3H-labeled roZogy 33, 513 (1967). proteins decline in proteins to W-labeled 3. RUSSELL, W. C., and BECICER, Y., Virology 35, value as the protein size increases and the 18 (1968). (ii) amino acid deprivation causes a marked 1. GOLDBLUM. N.. RAVID. Z.. and BECKER. Y.. decrease in the 3H/14C ratio of several bands. J. Gen. kiroiogy 3, 143 (i968). ’ ’ From the coincidence of position of valleys 6. TANKERSLEY, R. W., JR., J. Bacterial. 87, 609 signifying a decrease in 3H/14C ratios (Fig. 2) (1964). 6. JENEY, E., GONCZOL, E., and VACZ~, L., Acta with certain of the bands present in the gels Microbial. Acad. Sci. Hung. 14, 31 (1967). (Fig. l), we conclude that the proteins in at 7. INGLIS, V. B. M., J. Gen. Virology 3, 9 (1968). least five bands (with approximate molecular 8. ROIZMAN, B., SPRING, S. B. and ROANE, P. R., weights of 30,000, 45,000, 55,000, 100,000, JR., J. Virology 1, 181 (1967). and 120,000 daltons) are rich in arginine. 9. BECKER, Y., OLSHEVSKY, V., and LEVITT, J., One interpretation of the data is that the J. Gen. Virology 1, 471 (1967). inconstancy of 3H/14C ratio is due to accu10. PIEZ, K. A., and EAGLE, H., J. Biol. Chem. 231, mulation of fragments of large proteins in 533 (1958). il. GONCZOL, E., JENEY, E., and VACZI, L., Acta arginine-deprived cells. We conclude that Microbial. Acad. Sci. Hung. 14, 39 (1967). the contribution of fragments is trivial be12. SPEAR, P. G., and ROIZMAN, B., Virology 36, cause as shown in Fig. 1 (i) new bands con545 (1968) taining 3H peptides alone are absent, (ii) the is. SHIMONO, H., BEN-P• RAT, T., and KAPLAN, 3H peptides are not uniformly distributed in A. S., Virology 37, 49 (1969). the region of relatively small proteins, (iii) R. J., and ROIZMAN, B., Science 153, 14 SYDISICIS, the bands containing 3H or 14C peptides coin-76 (1966). cide, and (iv) the relative amount of 3H 16. SYDISKIS, R. J., and ROIZMAN, B., Virology 32, 678 (1968). counts between bands is not higher than 16. SPEAR, P. G., and ROIZMAN, B. Anal. Biowould be expected on the basis of 3H counts them. 26, 197 (1968). in the bands and from the distribution of 17. SHAPIRO, A. L., VINUELA, E., and MAIZEL, J. 1% counts in the gel. Since it is unlikely that V., Jr., Biochem. Biophys. Res. Commun. fragments would invariably coincide in size 5, 815 (1967). with small proteins made in control cultures we conclude that the inconstancy of the SUSAN B. SPRINGY 3H/14C ratio is not due to excessive accumuBERNARD ROIZMAN PATRICIA G. SPEARS lation of fragments. Our interpretation of the data presented in this paper and those Department of Microbiology cited earlier is the following: (i) The argiThe University of Chicago nine pool present in cells infected at the onset of arginine deprivation is sufficient to Chicago, Illinois 60657 allow the synthesis of proteins necessary for Accepted May 26, 1969 the initiation of the biosynthesis of viral 2 S. B. Spring was a postdoctoral trainee U. S. DNA. (ii) Once the arginine concentration Public Health Service (AI-90238~96). becomes limiting, the probability that a 3 P. G. Spear holds a U. S. Public Health Service given protein will be completed depends ou predoctoral fellowship (FlGM35 298-03).
SHORT
COMMUNICATIONS
14.
JARRETT, 0. and MACPHERSON, I. Intern. J. Cancer 3, 654662 (1968). 16. FAWCETT, D. W. An Atlas of Fine Structure. Saunders, Philadelphia, Pennsylvania (1966). A. F. Cancer Res. 16. DALES, S. and HOWATSON, 21, 193-197 (1961). 17. KINDIG, D. A. and KIRSTEN, W. H. Science 155, 1543-1545 (1967). 18. CROMACK, A. S. J. Gen. Viral. 2, 195198 (1968). 19. MUSSGAY, M., RECZKO, E. and AHL, R. J. Gen. Viral. 4, 445447 (1969). CHARLES SHIPMAN, JR. GRETCHEN C. VANDER WEIDE BOOE IL MA~ Department of Oral Riology School of Dentistry and Department of Microbiology School of Medicine The University of Michigan Ann Arbor, Michigan .@lOJ Accepted
May
RO, 1969
1 Public DE42829-01).
Health
Special
Selective
Failure
of Protein
Herpesvirus-Infected
Fellow
Cells
(No.
Synthesis
F03
in
Deprived
of Arginine’ The yield of several DNA viruses (l-4), including that of herpes simplex virus (5-8), from animal cells in continuous cultivation is largely dependent on the presence of arginine in the medium. The requirement for arginine is more stringent than that for other amino acids (5) but it is not uniform; the yield of herpes simplex virus from primary cultures of human embryonic fibroblasts, chick embryo fibroblasts, or of monkey kidney cells is unaffected by arginine deprivation (6). The differences between primary and continuous cell cultures with respect to the capacity to support virus multiplication in arginine-free medium may reflect the size of the arginine pool (9-11) . Arginine starvation of cells in continuous cultivation does not 1 Aided by grants from the National Cancer Institute, U. S. Public Health Service (CA O&194), the American Cancer Society (E 314E), and the National Science Foundation (GB 8242).
prevent adsorption, penetration, uncoating of herpes simplex virus, and reproductive events occurring during the first several hours after infection. The latter include the synthesis of viral DNA (9) and the synthesis of viral proteins and their transport from cytoplasm into the nucleus as evidenced by the presence of viral antigens in both compartments of the cell (8). However, immunofluorescent granules in the cytoplasm and nucleus and viral particles are not made (8). These findings suggest that arginine deprivation causes a selective decrease in the synthesis of some viral macromolecules. One hypothesis that could explain these findings is based on the assumption that in infected cells depleted of arginine the translation of mRNA comes to a halt at codons specifying arginine. It could be expected therefore that in arginine-depleted cells the probability that a given peptide is completed would be inversely proportional to its molecular weight and to its arginine content. The hypothesis is of particular interest in view of the evidence presented by us and others that herpesvirus contains proteins ranging up to 125,000 daltons (12, 1s). To test this hypothesis we compared the size distribution of proteins made in untreated and arginine-deprived infected cells. In these experiments replicate HEp-2 cell cultures, each containing 2 X lo7 cells, were exposed for 1 hour at 37” to sufficient virus to yield a multiplicity of 50 plaque-forming units per cell. One set of cultures (control) was then overlaid with mixture 199 containing 1% dialyzed calf serum (199-I). The other set was overlaid with the same medium but lacking arginine (199-1 urg-). At 6 hours postinfection the maintenance medium was decanted, the cells were washed, and replenished as follows: Control cultures received medium 199-I lacking lysine (199-I Zys-) but supplemented with 0.2 PCi of 14C-l-lysine per ml of medium. The arginine-deprived cultures were replenished with medium 199-1 arg-Lys supplemented with 2 &i of 3H-llysine per ml of medium. The final concentration of lysine in the two sets was approximately the same. The cells were then reincubated. The time of labeling was based on the observations that host polyribosomes
SHORT
COMMUNICATIONS
o+-
01 d...,.........r.r
\
711
deprived infected cells were layered on 5.7 % acrylamide gels 20 cm long and electrophoresed for 18 hours. The gels were then sliced and radioactivity measured as previously described (16). To discriminate between the effects of arginine deprivation and the effects of partial lysine deprivation necessary to label the proteins, we repeated the experiments twice. In the second experiment we used media 199-l arg-leu- and 199-1 i&u- supplemented with 3H-l-leucine and 14C-l-leucine, respectively. In the third experiment we used 199-l arg- isoleu- and 199-l isoleu- supplemented with 3H-l-isoleu and 14C-1-isoleu, respectively. The choice of isoleucine, leucine, and lysine was predicated on the report by Tankersley (5) showing that the deprivation of these amino 1 1EUClNE
FIG. 1. Electrophoretic profiles of artificial mixtures of (i) proteins labeled with single 3H amino acids (either lysine, leucine, or isoleucine) in herpes simplex virus-infected HEp-2 cells deprived of arginine, and (ii) proteins labeled with the corresponding W amino acid in untreated infected cells. The cells were deprived of arginine immediately after the l-hour exposure to virus. The proteins were labeled between 6 and 20 hours after infection. Previous studies had demonstrated that host protein synthesis ceases at 4 hours postinfection (12, 14, 15). The procedures for preparation of the labeled cell lysate, electrophoresis, and countingof radioactivity in the gel slices were the same as previously described (12). The profile of radioactivity obtained with radioactive leucine is the same as previously described (12).
become disaggregated and proteins characteristic of uninfected cells ceaseto be made within 4 hours after infection (l&14,15). At 20 hours postinfection the cells were allowed to swell in distilled water and broken by 20 strokes of a tight Dounce homogenizer. The entire lysate was then solubilized in urea, sodium dodecyl sulfate, and p mercaptoethanol as previously described (12). Artificial mixtures of lysates of control and arginine-
FIG. 2. The ratio of 3H and 1% proteins in each gel slice computed from the profiles shown in Fig. 1 plotted as a function of the molecular weight of the protein in the gel. The molecular weights of proteins in the gel were estimated by co-electrophoresis of proteins made in infected cells with proteins of known size (12). For the gels used in this study there is a linear relationship between protein molecular weight and distance of migration plotted on a log scale (17).
712
SHORT
COMMUNICATIONS
acids has only a slight effect on virus yield. the number of arginine residues (i.e., given The profiles of radioactivity in the acrylathe same arginine frequency, shorter promide gels are shown in Fig. 1. Figure 2 shows teins have a higher probability of being comthe 3H/14C ratios computed from 3H and 14C pleted). (iii) Arginine deprivation may be more detrimental to virus multiplication counts of each gel slice and plotted as a function of the molecular weight of the proteins in than that of other amino acids because sevthe gels as determined by co-electrophoresis eral proteins specified by the virus are both large in size and rich in arginine. with proteins of known size (12, 17). It would be expected that if amino acid deprivation REFERENCES caused a decrease in the synthesis of all 1. ROUSE, H. C., BONIFAS, V. H., and proteins the 3H/14C ratio would remain relaSCHLDSINGER, R. W., Virology 30,357 (1963). tively constant. The data summarized in 2. ROUSE, H. C., and SCHLESINGER, R. W., ViFig. 2 show that (i) the ratios of 3H-labeled roZogy 33, 513 (1967). proteins decline in proteins to W-labeled 3. RUSSELL, W. C., and BECICER, Y., Virology 35, value as the protein size increases and the 18 (1968). (ii) amino acid deprivation causes a marked 1. GOLDBLUM. N.. RAVID. Z.. and BECKER. Y.. decrease in the 3H/14C ratio of several bands. J. Gen. kiroiogy 3, 143 (i968). ’ ’ From the coincidence of position of valleys 6. TANKERSLEY, R. W., JR., J. Bacterial. 87, 609 signifying a decrease in 3H/14C ratios (Fig. 2) (1964). 6. JENEY, E., GONCZOL, E., and VACZ~, L., Acta with certain of the bands present in the gels Microbial. Acad. Sci. Hung. 14, 31 (1967). (Fig. l), we conclude that the proteins in at 7. INGLIS, V. B. M., J. Gen. Virology 3, 9 (1968). least five bands (with approximate molecular 8. ROIZMAN, B., SPRING, S. B. and ROANE, P. R., weights of 30,000, 45,000, 55,000, 100,000, JR., J. Virology 1, 181 (1967). and 120,000 daltons) are rich in arginine. 9. BECKER, Y., OLSHEVSKY, V., and LEVITT, J., One interpretation of the data is that the J. Gen. Virology 1, 471 (1967). inconstancy of 3H/14C ratio is due to accu10. PIEZ, K. A., and EAGLE, H., J. Biol. Chem. 231, mulation of fragments of large proteins in 533 (1958). il. GONCZOL, E., JENEY, E., and VACZI, L., Acta arginine-deprived cells. We conclude that Microbial. Acad. Sci. Hung. 14, 39 (1967). the contribution of fragments is trivial be12. SPEAR, P. G., and ROIZMAN, B., Virology 36, cause as shown in Fig. 1 (i) new bands con545 (1968) taining 3H peptides alone are absent, (ii) the is. SHIMONO, H., BEN-P• RAT, T., and KAPLAN, 3H peptides are not uniformly distributed in A. S., Virology 37, 49 (1969). the region of relatively small proteins, (iii) R. J., and ROIZMAN, B., Science 153, 14 SYDISICIS, the bands containing 3H or 14C peptides coin-76 (1966). cide, and (iv) the relative amount of 3H 16. SYDISKIS, R. J., and ROIZMAN, B., Virology 32, 678 (1968). counts between bands is not higher than 16. SPEAR, P. G., and ROIZMAN, B. Anal. Biowould be expected on the basis of 3H counts them. 26, 197 (1968). in the bands and from the distribution of 17. SHAPIRO, A. L., VINUELA, E., and MAIZEL, J. 1% counts in the gel. Since it is unlikely that V., Jr., Biochem. Biophys. Res. Commun. fragments would invariably coincide in size 5, 815 (1967). with small proteins made in control cultures we conclude that the inconstancy of the SUSAN B. SPRINGY 3H/14C ratio is not due to excessive accumuBERNARD ROIZMAN PATRICIA G. SPEARS lation of fragments. Our interpretation of the data presented in this paper and those Department of Microbiology cited earlier is the following: (i) The argiThe University of Chicago nine pool present in cells infected at the onset of arginine deprivation is sufficient to Chicago, Illinois 60657 allow the synthesis of proteins necessary for Accepted May 26, 1969 the initiation of the biosynthesis of viral 2 S. B. Spring was a postdoctoral trainee U. S. DNA. (ii) Once the arginine concentration Public Health Service (AI-90238~96). becomes limiting, the probability that a 3 P. G. Spear holds a U. S. Public Health Service given protein will be completed depends ou predoctoral fellowship (FlGM35 298-03).