- Email: [email protected]
A comparison of transfer RNA from untreated and interferon-treated murine cells
VIROLOGY
74, 262-264
A Comparison
CLARENCE
(1976)
of Transfer
COLBY,’
EDWARD
RNA from Untreated Murine Cells E. PENHOET,2
AND
and Interferon-Treated
CHARLES
E. SAMUEL3p4
’ Department of Genetics, University of California, Davis, Calfornia 95616; ” Department of Biochemistry, University of California, Berkeley, California 94720; ” Section of Biochemistry and Molecular Biology, Department of Biological Sciences, University of California, Santa Barbara, California 93106 Accepted
July
14,1976
Transfer RNA isolated from untreated and from interferon-treated mouse L tibroblast and mouse ascites cells possess the same number and relative amounts of leucyl-tRNA isoacceptor species as identified by reversed-phase 5 chromatography and have comparable stimulatory activity in the reversal of the interferon-mediated inhibition of reovirus mRNA translation in vitro.
It is well established that interferon treatment of many species of mammalian cells in culture greatly reduces the ability of the treated cells to support the replication of a wide variety of DNA and RNA viruses (3). Several lines of evidence indicate that the interferon-mediated inhibition of virus multiplication is due, at least in part, to inhibition of translation of viral messenger RNA ( 7). Specifically, cell-free extracts prepared from interferon-treated murine cells catalyze the translation in vitro of exogenously added messenger RNA much less efficiently than extracts prepared from untreated cells (2,4,5,10). The interferon-mediated inhibition of viral mRNA translation appears to be facilitated by a transient ribosome-associated polypeptide factor which can be separated from ribosomes by either washing with buffers containing concentrated salt (2, 10) or incubation (5). Content et al. (I) and Gupta et al. (6) have reported that the interferon-mediated inhibition of translation observed in vitro can be reversed by the addition of transfer RNA isolated from either untreated control or interferontreated cells to protein synthesis systems prepared from interferon-treated cells. Hemoglobin mRNA and mengo RNA translation have been shown to be restored by ’ Author addressed.
to whom
requests
for reprints
should
different tRNA fractions; benzoylated DEAE-cellulose column chromatography fractions which were most active in restoring translation of mengo virus RNA showed significant aminoacylation only ,with leucine among 16 amino acids tested (I 1. Recently, the time course of aminoacylation of endogenous tRNA with all 20 amino acids catalyzed by Sephadextreated extracts from untreated and interferon-treated Ehrlich ascites cells was examined; the level of aminoacylation of leutine-specific tRNA was found to be decreased and the rate of inactivation of leucyl-tRNA increased as a result of interferon treatment (11). Because of experiments of the above nature, as part of our study of the molecular biology of the interferon system, we have performed experiments designed to compare both the chromatographic properties and protein synthesis stimulating activities of transfer RNA isolated from untreated and from interferon-treated murine cells. We have examined the isoacceptor leucyl-tRNA species present in mouse L fibroblast cells by chromatography of the tRNA aminoacylated in vivo on reversed-phase chromatography (RPC-5) columns. Figure 1 presents a comparison of leucyl-tRNA isolated from untreated control L cells and from L cells treated with mouse interferon. The resolving power of the RPC-5 chroma-
be 262
Copyright All riehts
0 1976 by Academic Press, Inc. of reumduction in anv form reserved.
SHORT
263
COMMUNICATIONS
40
Fraction
number
FIG. 1. Mouse L cells were grown in loo-mm tissue culture dishes in Dulbecco’s modified Eagle’s medium (DME) containing 10% calf serum. Interferon was prepared from Newcastle disease virus infected L cells by the method of Lampson et al. (8). Replicate cultures were treated with 10 units of interferon for 18 hr in DME and 10% CS. To obtain radioactively labeled leucyl tRNA in u&o, cultures were washed twice with phosphate-buffered saline (PBS) at 37” and then incubated for 5 min in leucine-free minimal essential medium (MEM); the medium was then replaced with 2 ml of the same medium containing radioactive leucine (20 &i of [Wlleucine or 200 &i of [3Hlleucine). The cultures were incubated in 13Hl- or [“Clleucine containing medium for 5 min after which time the medium was removed and the cells were washed at 4 with 50 mM NaOAc buffer, pH 5.2. The cells were transferred to a test tube containing 4 ml of phenol saturated with 50 mM NaOAc buffer, pH 5.2. The suspension was agitated vigorously for 3 min and then centrifuged at 1500g for 10 min. The aqueous supernatant was removed and combined with two volumes of 95% ethanol and stored at -20”. The flocculent precipitate which formed was collected by centrifugation, washed, and dissolved in approximately 0.2 ml of chromatography buffer (10 mM NaOAc, 10 mM MgCl,, 2 mM 2-mercaptoethanol). A suitable pair of 3H- and “C-labeled samples was then mixed and applied to a 0.6x 24-cm RPC-5 column at an operating pressure of 300 lb/in.’ (9). The column was washed with 40 ml of chromatography buffer containing 0.4 M NaCl and the tRNA was eluted with a linear 250 ml NaCl gradient (0.55-1.0 M) followed by a linear 90 ml NaCl gradient (1.0-1.5 M). Two-milliliter fractions were collected, transferred to scintillation counting vials, and diluted in 10 ml of Triton X-100 toluene-based scintillation fluid. The samples were counted in a Nuclear Chicago counter with settings appropriate for dual-label counting. ----- [‘Clleucyl-tRNA Iru from untreated L cells; ~ l”Hlleucyl-tRNA’t’” from interferon-treated L cells. Relative radioactivity: “C, 934 cpm - 1.0; 3H, 8820 cpm = 1.0.
tography system was suffkient to facilitate the fractionation of L cell tRNA into several different leucine isoaccepting tRNA. However, the leucyl-tRNA profiles obtained from tRNA prepared from untreated and from interferon-treated cells were indistinguishable (Fig. 1). We also performed analogous comparisons on virus-infected untreated and interferontreated L cells; again, the chromatographic profiles were virtually identical. In the course of our studies, we examined tRNA isolated from untreated L cells, vaccinia-infected cells, VSV-infected cells, interferon-treated cells, and various combinations of these. In no instance did treatment of mouse L fibroblast cells with interferon or infection of the cells with virus or a combination of both interferon-treatment and virus infection result in a discernible alteration in the RPC-5 column
chromatography elution profile of leucyltRNA species. Thus, neither the total leutine acceptance of unfractionated tRNA isolated directly from interferon-treated or control cells (11) nor the relative ratios of the fractionated leucine isoacceptor tRNA subspecies (Fig. 1) appear to be altered as a result of interferon treatment. We characterized the functional capacities of tRNA preparations from untreated and interferon-treated cells by examining their abilities to affect the translation of reovirus mRNA catalyzed by cell-free protein synthesizing systems prepared from untreated and interferon-treated ascites tumor cells. As shown in Fig. 2, exogenously added transfer RNA partially reverses the interferon-mediated inhibition of viral mRNA translation as previously reported by Content et al. (1) and Gupta et al. (6). However, the specific stimulatory
264
SHORT
20.
(C) YEAST
o
4
8
id)
COMMUNICATIONS
mediated inhibition of viral mRNA translation in vitro can be at least partially overcome by the addition of homologous transfer RNA to the protein synthesizing system. However, our studies also indicate that: (i) the number and relative amounts of leucyl-tRNA species as identified by reversed-phase 5 chromatography are unchanged upon interferon treatment of mouse L cells; and (ii) the specific stimulatory activity of tRNA prepared from interferon-treated cells is comparable to tRNA prepared from untreated cells in the reversal of the interferon-mediated inhibition of reovirus mRNA translation in. vitro.
RAT LIVER
12 16 20 0 4 TRANSFER RNA (&50~1)
8
12
16
20
FIG. 2. Effect of exogenously added tRNA on the translation of reovirus mRNA catalyzed by cell-free systems prepared from untreated control and interferon-treated cells. Cell-free extracts and reovirus mRNA were prepared as previously described (10); mouse ascites tRNA was prepared as described by Yang and Novelli (12); rat. liver tRNA was from Grand Island Biological Co. and Bakers’ yeast tRNA from Calbiochem. The reaction mixture for measuring L-L3Hlleucine incorporation into 90” trichloroacetic acid-insoluble material contained ascites S-20 supernatant fraction, 0.6 ml/ml; 30 mM HEPES, pH 7.5; 96 n&f KCl; 4 mM Mg(OAc),; 1 mM DTT; 1 mM ATP; 0.2 mM GTP; 0.6 mM CTP; 10 m&f creatine phosphate; 0.2 mg/ml creatine phosphokinase; unlabeled amino acids, 0.075 mM; L-l:‘H]leucine, 50 &i/ml; reovirus mRNA, 100 pg/ml; and stripped unfractionated tRNA as indicated. Incubation was at 31” for 45 min. (0-O) Cell-free system from untreated cells; (A-A) cell-free system from interferon-treated cells. (a) Ascites tRNA from untreated cells; (b) ascites tRNA from interferontreated cells; (c)yeast tRNA; and (d) rat liver tRNA.
activity of tRNA prepared from interferontreated cells is comparable to that prepared from untreated cells. Yeast and rat liver tRNA are slightly inhibitory when tested in the untreated cell-free system and do not significantly stimulate reovirus mRNA translation catalyzed by the interferon-treated cell-free protein synthesizing system. In this study, we have confirmed the findings of Content et al. (1) and Gupta et al. (6) which indicate that the interferon-
This work was supported in part by Research Grants from the National Institute of Allergy and Infectious Diseases (AI-11886 and AI-125201, the National Science Foundation (GB-38658), and the American Cancer Society (VC-192). REFERENCES 1. CONTENT, J., LEBLEU, B., ZILBERSTEIN, A., BERISSI, H., and REVEL, M., FEBS Lett. 41, 125130 (1974). 2. FALCOFF, E., FALCOFF, R., LEBLEU, B., and REVEL, M., J. Viral. 12, 421-430 (1973). 3. FINTER, N. B., In “Frontiers of Biology,” Vol. 2. American Elsevier, New York, 1973. 4. FRIEDMAN, R. M., METZ, D. H., ESTEBAN, R. M., TAVELL, D. R., BALL, L. A., and KERR, I. M., J. Vi&. 10, 1184-1198 (1972). 5. GUPTA, S. L., SOPARI, M. L., and LENGYEL, P., Biochem. Biophys. Res. Commun. 54, 777-783 (1973). 6. GUPTA, S. L., SOPORI, M. L., and LENGYEL, P., Biochem. Biophys. Res. Commun. 57, 763-770 (1974). 7. Ho, M., and ARMSTRONG, J. A., Ann. Rev. Microbiol. 29, 131-161 (1975). 8. LAMPSON, G. P., TYTELL, A. A., NEMES, M. M., and HILLEMAN, M. R., Proc. Sot. Exp. Biol. Med. 112, 468-478 (1963). 9. PEARSON, R. L., WEISS, J. F., and KELMERS, A. D., Biochim. Biophys. Acta 228, 770-774 (1971). 10. SAMUEL, C. E., and JOKLIK, W. K., Virology 58, 476-491 (1974). 11. SEN, G. C., GU~TA, S. L., BROWN, G. E., LEBLEU, B., REBELU), M. A., and LENGYEL, P., J. Viral. 17, 191-203 (1976). 12. YANG, W. K., and NOVELLI, G. D., Methods in Enzymology 20, 44-55 (1971).
74, 262-264
A Comparison
CLARENCE
(1976)
of Transfer
COLBY,’
EDWARD
RNA from Untreated Murine Cells E. PENHOET,2
AND
and Interferon-Treated
CHARLES
E. SAMUEL3p4
’ Department of Genetics, University of California, Davis, Calfornia 95616; ” Department of Biochemistry, University of California, Berkeley, California 94720; ” Section of Biochemistry and Molecular Biology, Department of Biological Sciences, University of California, Santa Barbara, California 93106 Accepted
July
14,1976
Transfer RNA isolated from untreated and from interferon-treated mouse L tibroblast and mouse ascites cells possess the same number and relative amounts of leucyl-tRNA isoacceptor species as identified by reversed-phase 5 chromatography and have comparable stimulatory activity in the reversal of the interferon-mediated inhibition of reovirus mRNA translation in vitro.
It is well established that interferon treatment of many species of mammalian cells in culture greatly reduces the ability of the treated cells to support the replication of a wide variety of DNA and RNA viruses (3). Several lines of evidence indicate that the interferon-mediated inhibition of virus multiplication is due, at least in part, to inhibition of translation of viral messenger RNA ( 7). Specifically, cell-free extracts prepared from interferon-treated murine cells catalyze the translation in vitro of exogenously added messenger RNA much less efficiently than extracts prepared from untreated cells (2,4,5,10). The interferon-mediated inhibition of viral mRNA translation appears to be facilitated by a transient ribosome-associated polypeptide factor which can be separated from ribosomes by either washing with buffers containing concentrated salt (2, 10) or incubation (5). Content et al. (I) and Gupta et al. (6) have reported that the interferon-mediated inhibition of translation observed in vitro can be reversed by the addition of transfer RNA isolated from either untreated control or interferontreated cells to protein synthesis systems prepared from interferon-treated cells. Hemoglobin mRNA and mengo RNA translation have been shown to be restored by ’ Author addressed.
to whom
requests
for reprints
should
different tRNA fractions; benzoylated DEAE-cellulose column chromatography fractions which were most active in restoring translation of mengo virus RNA showed significant aminoacylation only ,with leucine among 16 amino acids tested (I 1. Recently, the time course of aminoacylation of endogenous tRNA with all 20 amino acids catalyzed by Sephadextreated extracts from untreated and interferon-treated Ehrlich ascites cells was examined; the level of aminoacylation of leutine-specific tRNA was found to be decreased and the rate of inactivation of leucyl-tRNA increased as a result of interferon treatment (11). Because of experiments of the above nature, as part of our study of the molecular biology of the interferon system, we have performed experiments designed to compare both the chromatographic properties and protein synthesis stimulating activities of transfer RNA isolated from untreated and from interferon-treated murine cells. We have examined the isoacceptor leucyl-tRNA species present in mouse L fibroblast cells by chromatography of the tRNA aminoacylated in vivo on reversed-phase chromatography (RPC-5) columns. Figure 1 presents a comparison of leucyl-tRNA isolated from untreated control L cells and from L cells treated with mouse interferon. The resolving power of the RPC-5 chroma-
be 262
Copyright All riehts
0 1976 by Academic Press, Inc. of reumduction in anv form reserved.
SHORT
263
COMMUNICATIONS
40
Fraction
number
FIG. 1. Mouse L cells were grown in loo-mm tissue culture dishes in Dulbecco’s modified Eagle’s medium (DME) containing 10% calf serum. Interferon was prepared from Newcastle disease virus infected L cells by the method of Lampson et al. (8). Replicate cultures were treated with 10 units of interferon for 18 hr in DME and 10% CS. To obtain radioactively labeled leucyl tRNA in u&o, cultures were washed twice with phosphate-buffered saline (PBS) at 37” and then incubated for 5 min in leucine-free minimal essential medium (MEM); the medium was then replaced with 2 ml of the same medium containing radioactive leucine (20 &i of [Wlleucine or 200 &i of [3Hlleucine). The cultures were incubated in 13Hl- or [“Clleucine containing medium for 5 min after which time the medium was removed and the cells were washed at 4 with 50 mM NaOAc buffer, pH 5.2. The cells were transferred to a test tube containing 4 ml of phenol saturated with 50 mM NaOAc buffer, pH 5.2. The suspension was agitated vigorously for 3 min and then centrifuged at 1500g for 10 min. The aqueous supernatant was removed and combined with two volumes of 95% ethanol and stored at -20”. The flocculent precipitate which formed was collected by centrifugation, washed, and dissolved in approximately 0.2 ml of chromatography buffer (10 mM NaOAc, 10 mM MgCl,, 2 mM 2-mercaptoethanol). A suitable pair of 3H- and “C-labeled samples was then mixed and applied to a 0.6x 24-cm RPC-5 column at an operating pressure of 300 lb/in.’ (9). The column was washed with 40 ml of chromatography buffer containing 0.4 M NaCl and the tRNA was eluted with a linear 250 ml NaCl gradient (0.55-1.0 M) followed by a linear 90 ml NaCl gradient (1.0-1.5 M). Two-milliliter fractions were collected, transferred to scintillation counting vials, and diluted in 10 ml of Triton X-100 toluene-based scintillation fluid. The samples were counted in a Nuclear Chicago counter with settings appropriate for dual-label counting. ----- [‘Clleucyl-tRNA Iru from untreated L cells; ~ l”Hlleucyl-tRNA’t’” from interferon-treated L cells. Relative radioactivity: “C, 934 cpm - 1.0; 3H, 8820 cpm = 1.0.
tography system was suffkient to facilitate the fractionation of L cell tRNA into several different leucine isoaccepting tRNA. However, the leucyl-tRNA profiles obtained from tRNA prepared from untreated and from interferon-treated cells were indistinguishable (Fig. 1). We also performed analogous comparisons on virus-infected untreated and interferontreated L cells; again, the chromatographic profiles were virtually identical. In the course of our studies, we examined tRNA isolated from untreated L cells, vaccinia-infected cells, VSV-infected cells, interferon-treated cells, and various combinations of these. In no instance did treatment of mouse L fibroblast cells with interferon or infection of the cells with virus or a combination of both interferon-treatment and virus infection result in a discernible alteration in the RPC-5 column
chromatography elution profile of leucyltRNA species. Thus, neither the total leutine acceptance of unfractionated tRNA isolated directly from interferon-treated or control cells (11) nor the relative ratios of the fractionated leucine isoacceptor tRNA subspecies (Fig. 1) appear to be altered as a result of interferon treatment. We characterized the functional capacities of tRNA preparations from untreated and interferon-treated cells by examining their abilities to affect the translation of reovirus mRNA catalyzed by cell-free protein synthesizing systems prepared from untreated and interferon-treated ascites tumor cells. As shown in Fig. 2, exogenously added transfer RNA partially reverses the interferon-mediated inhibition of viral mRNA translation as previously reported by Content et al. (1) and Gupta et al. (6). However, the specific stimulatory
264
SHORT
20.
(C) YEAST
o
4
8
id)
COMMUNICATIONS
mediated inhibition of viral mRNA translation in vitro can be at least partially overcome by the addition of homologous transfer RNA to the protein synthesizing system. However, our studies also indicate that: (i) the number and relative amounts of leucyl-tRNA species as identified by reversed-phase 5 chromatography are unchanged upon interferon treatment of mouse L cells; and (ii) the specific stimulatory activity of tRNA prepared from interferon-treated cells is comparable to tRNA prepared from untreated cells in the reversal of the interferon-mediated inhibition of reovirus mRNA translation in. vitro.
RAT LIVER
12 16 20 0 4 TRANSFER RNA (&50~1)
8
12
16
20
FIG. 2. Effect of exogenously added tRNA on the translation of reovirus mRNA catalyzed by cell-free systems prepared from untreated control and interferon-treated cells. Cell-free extracts and reovirus mRNA were prepared as previously described (10); mouse ascites tRNA was prepared as described by Yang and Novelli (12); rat. liver tRNA was from Grand Island Biological Co. and Bakers’ yeast tRNA from Calbiochem. The reaction mixture for measuring L-L3Hlleucine incorporation into 90” trichloroacetic acid-insoluble material contained ascites S-20 supernatant fraction, 0.6 ml/ml; 30 mM HEPES, pH 7.5; 96 n&f KCl; 4 mM Mg(OAc),; 1 mM DTT; 1 mM ATP; 0.2 mM GTP; 0.6 mM CTP; 10 m&f creatine phosphate; 0.2 mg/ml creatine phosphokinase; unlabeled amino acids, 0.075 mM; L-l:‘H]leucine, 50 &i/ml; reovirus mRNA, 100 pg/ml; and stripped unfractionated tRNA as indicated. Incubation was at 31” for 45 min. (0-O) Cell-free system from untreated cells; (A-A) cell-free system from interferon-treated cells. (a) Ascites tRNA from untreated cells; (b) ascites tRNA from interferontreated cells; (c)yeast tRNA; and (d) rat liver tRNA.
activity of tRNA prepared from interferontreated cells is comparable to that prepared from untreated cells. Yeast and rat liver tRNA are slightly inhibitory when tested in the untreated cell-free system and do not significantly stimulate reovirus mRNA translation catalyzed by the interferon-treated cell-free protein synthesizing system. In this study, we have confirmed the findings of Content et al. (1) and Gupta et al. (6) which indicate that the interferon-
This work was supported in part by Research Grants from the National Institute of Allergy and Infectious Diseases (AI-11886 and AI-125201, the National Science Foundation (GB-38658), and the American Cancer Society (VC-192). REFERENCES 1. CONTENT, J., LEBLEU, B., ZILBERSTEIN, A., BERISSI, H., and REVEL, M., FEBS Lett. 41, 125130 (1974). 2. FALCOFF, E., FALCOFF, R., LEBLEU, B., and REVEL, M., J. Viral. 12, 421-430 (1973). 3. FINTER, N. B., In “Frontiers of Biology,” Vol. 2. American Elsevier, New York, 1973. 4. FRIEDMAN, R. M., METZ, D. H., ESTEBAN, R. M., TAVELL, D. R., BALL, L. A., and KERR, I. M., J. Vi&. 10, 1184-1198 (1972). 5. GUPTA, S. L., SOPARI, M. L., and LENGYEL, P., Biochem. Biophys. Res. Commun. 54, 777-783 (1973). 6. GUPTA, S. L., SOPORI, M. L., and LENGYEL, P., Biochem. Biophys. Res. Commun. 57, 763-770 (1974). 7. Ho, M., and ARMSTRONG, J. A., Ann. Rev. Microbiol. 29, 131-161 (1975). 8. LAMPSON, G. P., TYTELL, A. A., NEMES, M. M., and HILLEMAN, M. R., Proc. Sot. Exp. Biol. Med. 112, 468-478 (1963). 9. PEARSON, R. L., WEISS, J. F., and KELMERS, A. D., Biochim. Biophys. Acta 228, 770-774 (1971). 10. SAMUEL, C. E., and JOKLIK, W. K., Virology 58, 476-491 (1974). 11. SEN, G. C., GU~TA, S. L., BROWN, G. E., LEBLEU, B., REBELU), M. A., and LENGYEL, P., J. Viral. 17, 191-203 (1976). 12. YANG, W. K., and NOVELLI, G. D., Methods in Enzymology 20, 44-55 (1971).