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Failure of globin mRNA to stimulate globin synthesis in cell-free extracts of interferon-treated globin-synthesizing mouse erythroleukemic cells
VIROLQGY
69, 360-363
(19761
Failure of Globin mRNA to Stimulate Globin Synthesis Extracts of Interferon-Treated Globin-Synthesizing Erythroleukemic Cells G. HILLER, Znstitut fiir Virologie
I. WINKLER, und Zmmunbiologie
G. VIEHHAUSER, a’er Universitit
Wiinburg,
in Cell-Free Mouse
C. JUNGWIRTH Wiinburg,
West Germany
G. BODO Ernst Boehringer
Znstitut fiir Anneimittelforschung,
Vienna, Austria
AND
S. DUBE’
W. OSTERTAG
AND
Max Plnnck Znstitut fiir experimentelle Medizin, Gdttingen, West Germany Accepted September 3,1975 Exogenous globin mRNA or encephalomyocarditis virus mRNA can be efficiently translated by S30 lysates of mouse erythroleukemic cells and also by lysates of similar cells in which hemoglobin synthesis had been induced by dimethylsulfoxide. Cell-free extracts from interferon-treated cells had not only lost their translation capacity for viral but also for globin mRNA. The indiscriminate inhibition of the in vitro translation capacity of natural mRNA often seen in extracts from interferon-treated murine cells seems therefore to be caused by a defect of the cell-free systems. The defect in the S30 lysates of interferon-treated cells could be repaired by the addition of tRNA.
Treatment of cells with interferon induces an antiviral state that does not permit viral replication. Cellular functions on the other hand are much less affected by interferon treatment. An apparent exception to this rule was the observation that translation of exogenous mRNA including viral and globin mRNA was inhibited to the same extent in cell-free extracts of some murine cells treated with interferon (1,2). This led to the hypothesis that interferon, at least in these cells, might have a dual effect. Recently, however, it has been shown that cellular functions necessary for induced erythroid differentiation and globin synthesis are not affected by interferon in erythroleukemia cells although release of Friend virus from the cells was blocked ‘Present address: California Institute of Technology, Division of Biology, Pasadena, Calif. 360 Copyright 0 1976 by Academic Ross, Inc. All rights of reproduction in any form reserved.
effectively (3, 4). We therefore expected that a cell-free system of interferontreated erythroleukemia cells should still allow translation of exogenous globin mRNA. However, in this paper we show that translation of exogenous globin and encephalomyocarditis virus (EM0 mRNA by S30 lysates from uninduced as well as dimethylsulfoxide (DMSO)-induced hemoglobin-synthesizing erythroleukemia cells is inhibited by interferon treatment of the cells. S30 lysates from uninduced or differentiating erythroleukemic F4-N cells were prepared (5). The S30 lysate of these cells efficiently translates the mRNA of EMC and rabbit globin (Table 1). The optimal conditions for the translation of EMC and globin mRNA are comparable to those wi
361
SHORT COMMUNICATIONS TABLE 1 TRANSLATION OF VIRAL AND CELLULAR MRNA IN CELL-FREE EXTRACTS (S30) FROM ERYTHROLEUKEMIC CELUI: EFFECT OF INTERFERON" Cells
mRNA added
W-labeled amino acid incorporated (pmoles) Control
Interferon treated
Unstimulated
PolyW) Globin EMC
2.9 6.9 2.0
1.9 0.2 0.2
Stimulated
PolyW) Globin EMC
a.2 12.0 4.9
7.1 0 0.4
a F4-N paraproerythroblasts were grown at a density of 5 x W-2 x W/ml (5). Partially purified mouse interferon (600 units/ml) was added U2). New medium was added together with new interferon (600 U/ml, calculated for the total volume) for two further l-day intervals. At the third day of interferon treatment cells were collected. Viral assays were carried out by the spleen focus-forming assay or by measuring reverse transcriptase activity in the supernatant fluid (3). Interferon reduced virus release by two orders of magnitude. One day before the beginning of induction with DMSO, interferon was added to F4-N cultures. New medium with 1% DMSO and 600 U/ml of interferon (both in final volume) was added at day 0 to maintain a cell density of 5 x W/ml. New medium with 1% DMSO was added at day 1 and day 2 to adjust cell density to 1 x W/ml. Interferon was added at day 1 and day 2 at 600 U/ml. The cells were harvested at day 3 of DMSO exposure. Hemoglobin synthesis was indistinguishable in the interferon- and DMSO-treated cultures from that of the DMSO-treated controls @, 4). Ninety-seven percent of the virus release was inhibited by interferon treatment. Preincubated cell-free extracts (S30) were prepared essentially as described by Mathews and Korner (13). Amino acid incorporation was measured ([Wlleucine for globin and [Wlphenylalanine for EMC virus-specific proteins) in a reaction volume of 0.05 ml containing: unlabeled amino acids, 50 @f; 4 fl [“Clphenylalanine (477 mCi/mmole) or [Wlleucine (346 mCi/mmole); 3 pg of globin or EMC mRNA, 10 fig of poly(U1; 30 mM Tris-HCI, pH 7.5; a5 r&f KCI; 2-3 mh4 Mg*+; 7 mM merceptoethanol; 1 m&f ATP, 0.1 m&f GTP, 0.6 m&f CTP; 10 m&f creatine phosphate; creatine kinase, 0.2 mg/ml; and 160 Mg of protein of S30 lysate. The preparation of EMC and globin mRNA has been described (14, 15). In vitro polypeptide synthesis was measured at 37”. Incorpo-
TABLE
1 -Continued
ration was linear at least over 40 min. Numbers are picomoles of W-labeled amino acids incorporated into acid-insoluble material from which the no mRNA background has been subtracted.
thesis of a product corn&rating in sodium dodecyl sulfate (SDS)-a&amide-gel electrophoresis with authentic rabbit globin. Under the same conditions histone mRNA could not be translated efficiently in the S30 lysate of erythroleukemic cells although the same mRNA was active in the stimulation of polypeptide synthesis in reticulocyte lysates (8). Addition of interferon to F4-N cells inhibits Friend virus release of uninduced cells (3, 4). S30 lysates prepared from interferon-treated uninduced erythroleukemic cells translated poly(U1 with approximately the same efficiency as extracts from control cells (Table 1). The extracts were however, under various ionic conditions, inactive in the translation of EMC or globin mRNA. DMSO stimulates erythroid differentiation and hemoglobin synthesis in F4-N cells (5). Interferon, if added before and during induction prevents the release of the endogenous spleen focus-forming virus complex but permits stimulation of globin synthesis (3, 4). Cell-free extracts of interferon-treated hemoglobin-synthesizing F4-N cells were prepared. These extracts also do not translate exogenously added EMC or rabbit globin mRNA (Table 1). This shows that the inhibition of globin mRNA translation in extracts from interferon-treated cells is not a property of the globin mRNA but, rather, caused by a defect in the cellfree system. The amino acid incorporation activities of mixtures of S30 lysates from interferontreated and control cells were as high as the S30 of the control cells alone (Fig. 1). The lack of translational activity therefore does not seem to be due to a soluble inhibitor. It is also most likely not caused by an enhanced nucleolytic activity. These results also show that the defect in the extract from interferon-treated cells cannot be repaired by control extract. The translational activity of interferon-treated extracts could not be restored by the addition
362
SHORT COMMUNICATIONS
. Hb
I 1
8
12
(6
20
24
21)
32
J
ps pcdel”
FIG. 1. Translation of globin mRNA in mixtures of extracts prepared from interferon-treated and control erythroblasts. Cultivation of cells, treatment with interferon (600 units/ml), preparation of extracts and reaction conditions for the in vitro synthesis of globin are the same as described in Table 1. Incorporation of radioactive amino acids into globin was proportional at least up to 300 pg of protein per assay of extracts from control cells. Reaction mixtures contained 153 pg of S30 lysate from interferon-treated cells and increasing amounts of extracts from control cells (A- --A). Reaction mixtures contained a constant amount of S30 lysate (160 pg) from control extracts and increasing amounts of extracts from interferon-treated cells (mn ). Abscissa, increasing amounts of cell extract added; ordinate, PC]leucine incorporated into acid-insoluble material.
of ribosomal wash preparations from rabbit reticulocytes (Fig. 2). The translation defect of the cell-free extracts from interferon-treated mouse Lcells or ascites cells can be repaired by adding mammalian tRNA or by removing an inhibitory factor associated with the ribosomes (7, 9-11). Addition of tRNA to
FIG. 2. In vitro translation of globin and EMC mRNA by preincubated extracts from mouse erythroblasts in the presence of a rabbit reticulocyte ribosomal wash. Experimental conditions are the same as in Table 1. Reaction mixtures contained 320 pg of protein (control) or 305 pg protein (interferon-treated) of 530 lysate and increasing amounts of crude ribosomal wash from rabbit reticulocytes (16). Solid lines, extracts from control cells; broken lines, extracts from interferon-treated cells. Abscissa, amount of ribosomal wash added to the reaction mixture; ordinate, radioactive amino acid incorporated into polypeptides.
the reaction mixture restores the translation activity of lysates from interferontreated uninduced and DMSO-induced erythroleukemic cells (Fig. 3). Translation activity of the extracts is restored for EMC as well as globin mRNA. Experiments with fractionated tRNA are presently being carried out to see if different tRNA species restore the translation capacity for globin and EMC mRNA in extracts from interferon-treated cells. Unstimulated and DMSO-induced
SHORT
363
COMMUNICATIONS ACKNOWLEDGMENTS This work was supported Deutsche Forschungsgemeinschaft and W. Ostertag).
by
grants (C.
from the Jungwirth
REFERENCES 1. FALCOFF, E., FALCOFF, R., LEBLEU, B., and REVEL, M., Nature &on&n) 240,145147 (1972). 2. GUFTA, S. L., SOPORI, M. L., and LENGYEL, P.,
I-
B&hem. (1973). 3. SWETLY,
I-
Biophys. Res. Commun. 54,777-783
OSTERTAG, W., Nature &on(1974). LIEBERMAN, D., VOLOCH, Z., AVIV, H., NUDEL, V., and REVEL, M., Mol. Biol. Rep. 1,477-451 (1974). OSTERTAG, W., MELDERIS, H., STEINHEIDER, G., KLUGE, N., and DUBE, S. K., Nature New Bid. 239, 231-234 (1972). EGGEN, K. L., and SHATKIN, A. J., J. Virol. 9, 636-645 (1972). SAMUELS, C. E., and JOKLIK, W. K., Virology 58, 476-491 (1974). BREINDL, M., and GALLWITZ, D., Eur. J. Bb them. 32, 381-391 (1973). CONTENT, J., LEBLEU, B., ZILBERSTEIN, A., BERISSI, H., and REVEL, M., FEBS Lett. 41, 125130 (1974). GUPTA, S. L., Soporu, M. L., and LENGYEL, P., P., and
&n) 251, 642-644,
4.
5.
6. I.
7. I-
8. 9. ,
FIG. 3. Reversion of the translation inhibition in cell-free extracts from interferon-treated unstimulated cells by the addition of tRNA. Assay conditions for in vitro amino acid incorporation are the same as in Table 1. Abscissa, amount of unfractionated chick muscle tRNA added to the reaction mixture; ordinate, radioactive amino acid incorporated into polypeptides. Solid lines, extracts from control cells; broken lines, extract from interferon-treated (600 units/ml) cells. Essentially similar results were obtained with extracts from interferon-treated and DMSO-induced cells.
mouse erythroleukemic cells seem to be a useful system for study of the mechanism of action of interferon both in intact cells and on the subcellular level.
10.
B&hem. 11.
Biophys. Res. Commun. 57,763-770
(1974). GUFTA, S. L.,
B&hem.
SOPORI,
M. L., and
LENGYEL,
P.,
Biophys. Res. Commun. 54,777-783
(1973).
12. JUNGWIRTH, 13.
J., and (1972). MATHEWS,
C., HORAK, I., Bono, G., LINDNER, SCHULTZE, B., Virology 48, 59-70 M.
B., and KORNER,
them. 17, 328-338 (1970). 14. LINGREL, J. B., In “Methods 15. 16.
A.,
Eur. J. Bio-
in Molecular Biology” (J. A. Last and A. I. Laskin, eds.), Vol. 2, p. 231. Marcel Dekker, New York, 1972. KERR, J., COHEN, N., and WORK, T. S., Biochem. J. 98, 826-835 (1966). SHAFRITZ, D. A., and ANDERSON, W. F., J. Bid. Chem. 245, 5553-5559 (1970).
69, 360-363
(19761
Failure of Globin mRNA to Stimulate Globin Synthesis Extracts of Interferon-Treated Globin-Synthesizing Erythroleukemic Cells G. HILLER, Znstitut fiir Virologie
I. WINKLER, und Zmmunbiologie
G. VIEHHAUSER, a’er Universitit
Wiinburg,
in Cell-Free Mouse
C. JUNGWIRTH Wiinburg,
West Germany
G. BODO Ernst Boehringer
Znstitut fiir Anneimittelforschung,
Vienna, Austria
AND
S. DUBE’
W. OSTERTAG
AND
Max Plnnck Znstitut fiir experimentelle Medizin, Gdttingen, West Germany Accepted September 3,1975 Exogenous globin mRNA or encephalomyocarditis virus mRNA can be efficiently translated by S30 lysates of mouse erythroleukemic cells and also by lysates of similar cells in which hemoglobin synthesis had been induced by dimethylsulfoxide. Cell-free extracts from interferon-treated cells had not only lost their translation capacity for viral but also for globin mRNA. The indiscriminate inhibition of the in vitro translation capacity of natural mRNA often seen in extracts from interferon-treated murine cells seems therefore to be caused by a defect of the cell-free systems. The defect in the S30 lysates of interferon-treated cells could be repaired by the addition of tRNA.
Treatment of cells with interferon induces an antiviral state that does not permit viral replication. Cellular functions on the other hand are much less affected by interferon treatment. An apparent exception to this rule was the observation that translation of exogenous mRNA including viral and globin mRNA was inhibited to the same extent in cell-free extracts of some murine cells treated with interferon (1,2). This led to the hypothesis that interferon, at least in these cells, might have a dual effect. Recently, however, it has been shown that cellular functions necessary for induced erythroid differentiation and globin synthesis are not affected by interferon in erythroleukemia cells although release of Friend virus from the cells was blocked ‘Present address: California Institute of Technology, Division of Biology, Pasadena, Calif. 360 Copyright 0 1976 by Academic Ross, Inc. All rights of reproduction in any form reserved.
effectively (3, 4). We therefore expected that a cell-free system of interferontreated erythroleukemia cells should still allow translation of exogenous globin mRNA. However, in this paper we show that translation of exogenous globin and encephalomyocarditis virus (EM0 mRNA by S30 lysates from uninduced as well as dimethylsulfoxide (DMSO)-induced hemoglobin-synthesizing erythroleukemia cells is inhibited by interferon treatment of the cells. S30 lysates from uninduced or differentiating erythroleukemic F4-N cells were prepared (5). The S30 lysate of these cells efficiently translates the mRNA of EMC and rabbit globin (Table 1). The optimal conditions for the translation of EMC and globin mRNA are comparable to those wi
361
SHORT COMMUNICATIONS TABLE 1 TRANSLATION OF VIRAL AND CELLULAR MRNA IN CELL-FREE EXTRACTS (S30) FROM ERYTHROLEUKEMIC CELUI: EFFECT OF INTERFERON" Cells
mRNA added
W-labeled amino acid incorporated (pmoles) Control
Interferon treated
Unstimulated
PolyW) Globin EMC
2.9 6.9 2.0
1.9 0.2 0.2
Stimulated
PolyW) Globin EMC
a.2 12.0 4.9
7.1 0 0.4
a F4-N paraproerythroblasts were grown at a density of 5 x W-2 x W/ml (5). Partially purified mouse interferon (600 units/ml) was added U2). New medium was added together with new interferon (600 U/ml, calculated for the total volume) for two further l-day intervals. At the third day of interferon treatment cells were collected. Viral assays were carried out by the spleen focus-forming assay or by measuring reverse transcriptase activity in the supernatant fluid (3). Interferon reduced virus release by two orders of magnitude. One day before the beginning of induction with DMSO, interferon was added to F4-N cultures. New medium with 1% DMSO and 600 U/ml of interferon (both in final volume) was added at day 0 to maintain a cell density of 5 x W/ml. New medium with 1% DMSO was added at day 1 and day 2 to adjust cell density to 1 x W/ml. Interferon was added at day 1 and day 2 at 600 U/ml. The cells were harvested at day 3 of DMSO exposure. Hemoglobin synthesis was indistinguishable in the interferon- and DMSO-treated cultures from that of the DMSO-treated controls @, 4). Ninety-seven percent of the virus release was inhibited by interferon treatment. Preincubated cell-free extracts (S30) were prepared essentially as described by Mathews and Korner (13). Amino acid incorporation was measured ([Wlleucine for globin and [Wlphenylalanine for EMC virus-specific proteins) in a reaction volume of 0.05 ml containing: unlabeled amino acids, 50 @f; 4 fl [“Clphenylalanine (477 mCi/mmole) or [Wlleucine (346 mCi/mmole); 3 pg of globin or EMC mRNA, 10 fig of poly(U1; 30 mM Tris-HCI, pH 7.5; a5 r&f KCI; 2-3 mh4 Mg*+; 7 mM merceptoethanol; 1 m&f ATP, 0.1 m&f GTP, 0.6 m&f CTP; 10 m&f creatine phosphate; creatine kinase, 0.2 mg/ml; and 160 Mg of protein of S30 lysate. The preparation of EMC and globin mRNA has been described (14, 15). In vitro polypeptide synthesis was measured at 37”. Incorpo-
TABLE
1 -Continued
ration was linear at least over 40 min. Numbers are picomoles of W-labeled amino acids incorporated into acid-insoluble material from which the no mRNA background has been subtracted.
thesis of a product corn&rating in sodium dodecyl sulfate (SDS)-a&amide-gel electrophoresis with authentic rabbit globin. Under the same conditions histone mRNA could not be translated efficiently in the S30 lysate of erythroleukemic cells although the same mRNA was active in the stimulation of polypeptide synthesis in reticulocyte lysates (8). Addition of interferon to F4-N cells inhibits Friend virus release of uninduced cells (3, 4). S30 lysates prepared from interferon-treated uninduced erythroleukemic cells translated poly(U1 with approximately the same efficiency as extracts from control cells (Table 1). The extracts were however, under various ionic conditions, inactive in the translation of EMC or globin mRNA. DMSO stimulates erythroid differentiation and hemoglobin synthesis in F4-N cells (5). Interferon, if added before and during induction prevents the release of the endogenous spleen focus-forming virus complex but permits stimulation of globin synthesis (3, 4). Cell-free extracts of interferon-treated hemoglobin-synthesizing F4-N cells were prepared. These extracts also do not translate exogenously added EMC or rabbit globin mRNA (Table 1). This shows that the inhibition of globin mRNA translation in extracts from interferon-treated cells is not a property of the globin mRNA but, rather, caused by a defect in the cellfree system. The amino acid incorporation activities of mixtures of S30 lysates from interferontreated and control cells were as high as the S30 of the control cells alone (Fig. 1). The lack of translational activity therefore does not seem to be due to a soluble inhibitor. It is also most likely not caused by an enhanced nucleolytic activity. These results also show that the defect in the extract from interferon-treated cells cannot be repaired by control extract. The translational activity of interferon-treated extracts could not be restored by the addition
362
SHORT COMMUNICATIONS
. Hb
I 1
8
12
(6
20
24
21)
32
J
ps pcdel”
FIG. 1. Translation of globin mRNA in mixtures of extracts prepared from interferon-treated and control erythroblasts. Cultivation of cells, treatment with interferon (600 units/ml), preparation of extracts and reaction conditions for the in vitro synthesis of globin are the same as described in Table 1. Incorporation of radioactive amino acids into globin was proportional at least up to 300 pg of protein per assay of extracts from control cells. Reaction mixtures contained 153 pg of S30 lysate from interferon-treated cells and increasing amounts of extracts from control cells (A- --A). Reaction mixtures contained a constant amount of S30 lysate (160 pg) from control extracts and increasing amounts of extracts from interferon-treated cells (mn ). Abscissa, increasing amounts of cell extract added; ordinate, PC]leucine incorporated into acid-insoluble material.
of ribosomal wash preparations from rabbit reticulocytes (Fig. 2). The translation defect of the cell-free extracts from interferon-treated mouse Lcells or ascites cells can be repaired by adding mammalian tRNA or by removing an inhibitory factor associated with the ribosomes (7, 9-11). Addition of tRNA to
FIG. 2. In vitro translation of globin and EMC mRNA by preincubated extracts from mouse erythroblasts in the presence of a rabbit reticulocyte ribosomal wash. Experimental conditions are the same as in Table 1. Reaction mixtures contained 320 pg of protein (control) or 305 pg protein (interferon-treated) of 530 lysate and increasing amounts of crude ribosomal wash from rabbit reticulocytes (16). Solid lines, extracts from control cells; broken lines, extracts from interferon-treated cells. Abscissa, amount of ribosomal wash added to the reaction mixture; ordinate, radioactive amino acid incorporated into polypeptides.
the reaction mixture restores the translation activity of lysates from interferontreated uninduced and DMSO-induced erythroleukemic cells (Fig. 3). Translation activity of the extracts is restored for EMC as well as globin mRNA. Experiments with fractionated tRNA are presently being carried out to see if different tRNA species restore the translation capacity for globin and EMC mRNA in extracts from interferon-treated cells. Unstimulated and DMSO-induced
SHORT
363
COMMUNICATIONS ACKNOWLEDGMENTS This work was supported Deutsche Forschungsgemeinschaft and W. Ostertag).
by
grants (C.
from the Jungwirth
REFERENCES 1. FALCOFF, E., FALCOFF, R., LEBLEU, B., and REVEL, M., Nature &on&n) 240,145147 (1972). 2. GUFTA, S. L., SOPORI, M. L., and LENGYEL, P.,
I-
B&hem. (1973). 3. SWETLY,
I-
Biophys. Res. Commun. 54,777-783
OSTERTAG, W., Nature &on(1974). LIEBERMAN, D., VOLOCH, Z., AVIV, H., NUDEL, V., and REVEL, M., Mol. Biol. Rep. 1,477-451 (1974). OSTERTAG, W., MELDERIS, H., STEINHEIDER, G., KLUGE, N., and DUBE, S. K., Nature New Bid. 239, 231-234 (1972). EGGEN, K. L., and SHATKIN, A. J., J. Virol. 9, 636-645 (1972). SAMUELS, C. E., and JOKLIK, W. K., Virology 58, 476-491 (1974). BREINDL, M., and GALLWITZ, D., Eur. J. Bb them. 32, 381-391 (1973). CONTENT, J., LEBLEU, B., ZILBERSTEIN, A., BERISSI, H., and REVEL, M., FEBS Lett. 41, 125130 (1974). GUPTA, S. L., Soporu, M. L., and LENGYEL, P., P., and
&n) 251, 642-644,
4.
5.
6. I.
7. I-
8. 9. ,
FIG. 3. Reversion of the translation inhibition in cell-free extracts from interferon-treated unstimulated cells by the addition of tRNA. Assay conditions for in vitro amino acid incorporation are the same as in Table 1. Abscissa, amount of unfractionated chick muscle tRNA added to the reaction mixture; ordinate, radioactive amino acid incorporated into polypeptides. Solid lines, extracts from control cells; broken lines, extract from interferon-treated (600 units/ml) cells. Essentially similar results were obtained with extracts from interferon-treated and DMSO-induced cells.
mouse erythroleukemic cells seem to be a useful system for study of the mechanism of action of interferon both in intact cells and on the subcellular level.
10.
B&hem. 11.
Biophys. Res. Commun. 57,763-770
(1974). GUFTA, S. L.,
B&hem.
SOPORI,
M. L., and
LENGYEL,
P.,
Biophys. Res. Commun. 54,777-783
(1973).
12. JUNGWIRTH, 13.
J., and (1972). MATHEWS,
C., HORAK, I., Bono, G., LINDNER, SCHULTZE, B., Virology 48, 59-70 M.
B., and KORNER,
them. 17, 328-338 (1970). 14. LINGREL, J. B., In “Methods 15. 16.
A.,
Eur. J. Bio-
in Molecular Biology” (J. A. Last and A. I. Laskin, eds.), Vol. 2, p. 231. Marcel Dekker, New York, 1972. KERR, J., COHEN, N., and WORK, T. S., Biochem. J. 98, 826-835 (1966). SHAFRITZ, D. A., and ANDERSON, W. F., J. Bid. Chem. 245, 5553-5559 (1970).