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Activation of endogenous type c virus by amino acid alcohols
VIROLOGY
88,
194-196
Activation CEDRIC
(1978)
of Endogenous
W. LONG,’
WILLIAM
Type C Virus by Amino Acid Alcohols A. SUK,
AND
CHARLOTTE
GREENAWALT
Viral Oncology Program, Frederick Cancer Research Center, Frederick, Maryland
21701
Accepted March 31, 1978 Amino acid alcohols were examined for their ability to inhibit protein synthesis and induce endogenous type C virus from Kiiten sarcoma virus (KiSV)-transformed Balb/c mouse cells. Histidinol and tyrosinol, amino alcohols of hi&dine and tyrosine, were very effective short-term activators of virus. Both inducers were very efficient inhibitors of protein synthesis reducing C3H]leucine incorporation by more than 90% in 1 hr. Two other alcohols, valinol and methioninol, also reduced protein synthesis but gave low activation. The activated virus had the host range of the xenotropic Balb virus:2, and following removal of inducer, the activated state decayed rapidly.
Several chemicals which alter macromolecular synthesis activate endogenous type C genomes from mouse cells. It was initially found that the halogenated pyrimidines 5bromodeoxyuridine (BUdR) and 5-iododeoxyuridine (IUdR) activated virus from AKR and Balb/c cells (I, 2). Subsequently, it was discovered that while halogenated pyrimidines activated both xenotropic Balb virus:2 and the N-tropic Balb virus& inhibitors of protein synthesis such as cycloheximide activated only the xenotropic virus from a KiSV transformed Balb/c (K-Balb) cell line (3) through a derepression of virusspecific RNA which accumulated in treated cells (4). Recently it has been shown that L-canavanine, an analog of arginine, can activate xenotropic virus from K-Balb cells, possibly through incorporation and formation of abnormal proteins regulating virus expression (5, 6). Because of endogenous protein turnover, deprivation of single amino acids from mammalian cells results in an approximate 80% decrease in protein synthesis over an extended time period ( 7). In contrast, cells cultured in the presence of an amino acid alcohol are very rapidly depleted of a charged tRNA-amino acid by an inhibition of activation by amino acyl-tRNA synthetase, thereby causing an almost complete cessation of protein synthesis. L-Histidinol, ’ To whom
reprint
requests
should
for example, has been shown to be a potent inhibitor of protein synthesis in cultured mouse L-cells (8) and human HeLa cells (9). In the present study, several amino acid alcohols were examined for inhibition of protein synthesis and induction of type C virus from K-Balb mouse cells. Induction experiments were performed using a KiSV transformed Balb/3T3 celI (K-Balb 19) cloned from a line originally obtained from Dr. S. A. Aaronson (NCI). Amino acid analogs were obtained from the following sources: isoleucinol, leucinol, serinol, and alaninol, Vega-Fox Biochemicals, methioninol and valinol, Aldrich Chemicals, tyrosinol, Sigma Chemicals; histidinol, Calbiochem. The cells were grown to confluence in Eagle’s minimal essential medium (EMEM) containing 100 IU of penicillin, 100 pg/ml of streptomycin and 10% fetal calf serum; 2 to 3 days later the cells were trypsinized and plated at 1.8 x lo4 cells/cm’. After 24 hr medium was replaced with induction medium containing 0.1 pg/ml of dexamethasone to amplify induction (10). When the inducer was an amino acid analog, the analogous amino acid was omitted from EMEM, and dialyzed fetal calf serum was used. Following incubation in induction medium, the drug was removed and the cells were treated with 20 pg/ml of mitomycin C for 1 hr and plated onto normal rat kidney (NRK) monolayers for focus formation ( 6).
be addressed. 194
0042-6822/78/0881-0194$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
Of the several amino acid alcohols examined for ability to induce virus in K-Balb cells, histidinol and tyrosinol proved to be very effective (Table 1). Two other alcohols, valinol and methioninol, gave low but reproducible activation. Histidinol, tyrosiTABLE CHEMICAL Treatment
Cycloheximide IUdR Histidinol
Tyrosinol
vaIin01
Isoleucinol
Methioninol
Leucinol
Serinol
AIaninol
INDUCTION Concentration (g/ml) 25 30 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10
1 OF TYPE C VIRUS Fzz
276 245 476 35 4 0 401 564 471 25 21 3 0 0 0 2 1 0 39 1 0 0 0 0 0 0 4 3 0 0 1, 0, 0, 0,
gh?-
(122)
[3H]I+ucme mcorporation* (%)
(239)
3.0 N.D. 6.0
(435)
3.5
5.0
160.0
21.0
70.0
90.0
3 0 0 0
49.0
“A total of 5 x Id induced cells was plated into NRK monolayers seeded 24 hr earlier in medium containing 2 pg/mI of polybrene at 1 x Id cells per 6cm dish. Induction period was 5 hr, except for 3-hr treatments given in parentheses. b C3H]Leucine (2 pCi/mI), L-leucine [4,5-3H(N)], New England Nuclear, 5 Ci/mmol) incorporation (6) w&s measured during the last 60 min of a 6-hr treatment with 500 ag of each alcohol. Incorporation was aIso measured in a noninduced control. All values were determined in triplicate.
195
nol, and valinol were the most effective inhibitors of protein synthesis in this cell system, each reducing [3H]leucine incorporation more than 95% following a 5-hr treatment. Methioninol reduced incorporation by 80%, and the other alcohols produced marginal effects. Concentrations of 0.25 mM tyrosinol and 0.50 mM histidinol gave near maximum induction during a 5-hr induction period. With tyrosinol, the most effective inducer, as little as a 3-hr treatment gave near maximum activation. Treatment of cells with each alcohol for longer periods did not appreciably change the levels of induction. To determine if induction frequencies could be increased above that found for each alcohol separately, histidinol and tyrosinol were added to cultures simultaneously for 5 or 16 hr at concentrations that gave either maximum (500 pg/ml) or submaximal activation. Under these conditions, the levels of induction did not increase. Viability of cells treated with either analog remained high during a 16-hr treatment. The addition of L-tyrosine at 20 pg/ml and r,-histidine at 50 pg/ml reduced the activation by the corresponding alcohol more than 90% proving these to be functional analogs in this cell system. Treatment of K-Balb cells with amino acid alcohols did not induce virus capable of forming foci on Balb/c or NIH swiss monolayers. The near maximum virus induction obtained within 3 hr after addition of tyrosinol suggested a rapid inhibition of protein synthesis. Incorporation of [3H]leucine was measured during 30-min intervals after addition of each alcohol at 0.5, 2, 4, and 6 hr, and induction of virus was followed at 1, 2, 3, 4, and 6 hr by infectious center assay (Fig. 1). During the first 30 min of treatment [3H]leucine incorporation was reduced 95% by tyrosinol and 80% by histidinol. Following a 2-hr treatment with tyrosin01 30-40s of maximum virus activation occurred. Histidinol and cycloheximide produced somewhat lower levels, but by 4 hr of treatment, all three chemicals had elicited 70-80s of their maximum induction capacity. Decay of virus expression following induction with either histidinol and tyrosin01 was rapid. Cells were induced with either analog (500 pg/ml) for 16 hr, and at
196
SHORT
COMMUNICATIONS
FIG. 1. Relationship between protein synthesis and virus induction. Cultures were treated with cycloheximide (25 pg/mI), histidinol (596 ag/mI), or tyrosinol (566 ag/mI) for variable time periods, and C3H]leucine incorporation and virus activation were measured concomitantly in duplicate dishes at the indicated times. Following treatment with inducers, cultures were treated with mitomycin C and seeded onto NRK monolayers. The data are expressed as percentages of the maximum induction obtained with each chemical following a 6-hr treatment. Incorporation with histidin01 (O), tyrosinol or cycloheximide (A). Virus induction with tyrosinol (A), histidinol (O), or cycloheximide (0).
tion times. This may reflect an accumulation of viral RNA with longer induction times (4). Histidinol and tyrosinol very efficiently activate type C virus from K-Balb cells in a readily reversible fashion following replacement with corresponding amino acids. Since virus induction following treatment with chemicals is a short-term process, the regulatory mechanism must turn over rapidly. The observation that valinol effrciently blocks protein synthesis without appreciable activation of virus may indicate a secondary process necessary for induction other than simply inhibition of protein synthesis. Chemicals of this nature should aid in the elucidation of molecular events necessary for virus activation. ACKNOWLEDGMENTS We are indebted to Dr. Marco Rabinovitz for helpful discussions and Dr. R. V. Gilden for continued interest and support. This work was supported by the Virus Cancer Program, Contract No. NOl-CO-75380, National Cancer Institute, NIH, Bethesda, Maryland 20614. REFERENCES
intervals following removal of inducer, cells were treated with mitomycin C and plated onto NRK monolayers for focus formation. By 20 hr following removal of either analog the number of foci had decreased to less than 5% of control values found at the time of removal of inducer. Twenty-four hours following removal of histidinol, [3H]leucine incorporation returned to normal levels. Activation of virus in cultures treated for 3 hr with histidinol or tyrosinol(500 pg/ml) was completely inhibited when either actinomycin D (1.2 pg/ml) or cytosine arabinoside (10 pg/ml) was present showing a requirement for RNA and DNA synthesis during induction. Cultures induced with histidinol for 2,3, or 4 hr followed by treatment with actinomycin D or cytosine arabinoside for 3 hr showed a decreasing sensitivity to posttreatment with longer induc-
1. LOWY, D. R., ROWE, W. P., TEICH, 2. 3. 4.
5. 6. 7’.
8. 9.
10.
N., and HARTLEY, J., Science 174, 155-156 (1971). AARONSON, S. A., TODARO, G. J., and SCOLNICK, E., Science 174, 157-159 (1971). AARONSON, S. A., and DUNN, C., Science 183, 422-424 (1974). CABRADILLA, C. D., ROBBINS, K. C., and AARONSON, S. A., Proc. Nat. Acad. Sci. USA 73, 4541-4545 (1976). AKSAMIT, R., and LONG, C. W., Virology 78, 567-570 (1977). AXSAMIT, R. R., CHRISTENSEN, W. L., and LONG, C. W., Virology 83,138-149 (1977). VAUGHAN, M. H., PAWLOWSKI, P. J., and FORCHAANMER, J., Proc. Nat. Acad. Sci. USA 68, 2057-2961 (1971). WARRINGTON, R. C., WRATTEN, N., and HECHTMAN, R., J. Biol. Chem. 262, 5251-5257 (1977). VAUGHAN, M. H., and HANSEN, B. S., J. Biol. Chem. 248.7087-7096 (1973). DUNN, C. Y., AARONSON, S. A., and STEPHENSON, J. R., Virology 66.579-588 (1975).
88,
194-196
Activation CEDRIC
(1978)
of Endogenous
W. LONG,’
WILLIAM
Type C Virus by Amino Acid Alcohols A. SUK,
AND
CHARLOTTE
GREENAWALT
Viral Oncology Program, Frederick Cancer Research Center, Frederick, Maryland
21701
Accepted March 31, 1978 Amino acid alcohols were examined for their ability to inhibit protein synthesis and induce endogenous type C virus from Kiiten sarcoma virus (KiSV)-transformed Balb/c mouse cells. Histidinol and tyrosinol, amino alcohols of hi&dine and tyrosine, were very effective short-term activators of virus. Both inducers were very efficient inhibitors of protein synthesis reducing C3H]leucine incorporation by more than 90% in 1 hr. Two other alcohols, valinol and methioninol, also reduced protein synthesis but gave low activation. The activated virus had the host range of the xenotropic Balb virus:2, and following removal of inducer, the activated state decayed rapidly.
Several chemicals which alter macromolecular synthesis activate endogenous type C genomes from mouse cells. It was initially found that the halogenated pyrimidines 5bromodeoxyuridine (BUdR) and 5-iododeoxyuridine (IUdR) activated virus from AKR and Balb/c cells (I, 2). Subsequently, it was discovered that while halogenated pyrimidines activated both xenotropic Balb virus:2 and the N-tropic Balb virus& inhibitors of protein synthesis such as cycloheximide activated only the xenotropic virus from a KiSV transformed Balb/c (K-Balb) cell line (3) through a derepression of virusspecific RNA which accumulated in treated cells (4). Recently it has been shown that L-canavanine, an analog of arginine, can activate xenotropic virus from K-Balb cells, possibly through incorporation and formation of abnormal proteins regulating virus expression (5, 6). Because of endogenous protein turnover, deprivation of single amino acids from mammalian cells results in an approximate 80% decrease in protein synthesis over an extended time period ( 7). In contrast, cells cultured in the presence of an amino acid alcohol are very rapidly depleted of a charged tRNA-amino acid by an inhibition of activation by amino acyl-tRNA synthetase, thereby causing an almost complete cessation of protein synthesis. L-Histidinol, ’ To whom
reprint
requests
should
for example, has been shown to be a potent inhibitor of protein synthesis in cultured mouse L-cells (8) and human HeLa cells (9). In the present study, several amino acid alcohols were examined for inhibition of protein synthesis and induction of type C virus from K-Balb mouse cells. Induction experiments were performed using a KiSV transformed Balb/3T3 celI (K-Balb 19) cloned from a line originally obtained from Dr. S. A. Aaronson (NCI). Amino acid analogs were obtained from the following sources: isoleucinol, leucinol, serinol, and alaninol, Vega-Fox Biochemicals, methioninol and valinol, Aldrich Chemicals, tyrosinol, Sigma Chemicals; histidinol, Calbiochem. The cells were grown to confluence in Eagle’s minimal essential medium (EMEM) containing 100 IU of penicillin, 100 pg/ml of streptomycin and 10% fetal calf serum; 2 to 3 days later the cells were trypsinized and plated at 1.8 x lo4 cells/cm’. After 24 hr medium was replaced with induction medium containing 0.1 pg/ml of dexamethasone to amplify induction (10). When the inducer was an amino acid analog, the analogous amino acid was omitted from EMEM, and dialyzed fetal calf serum was used. Following incubation in induction medium, the drug was removed and the cells were treated with 20 pg/ml of mitomycin C for 1 hr and plated onto normal rat kidney (NRK) monolayers for focus formation ( 6).
be addressed. 194
0042-6822/78/0881-0194$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
Of the several amino acid alcohols examined for ability to induce virus in K-Balb cells, histidinol and tyrosinol proved to be very effective (Table 1). Two other alcohols, valinol and methioninol, gave low but reproducible activation. Histidinol, tyrosiTABLE CHEMICAL Treatment
Cycloheximide IUdR Histidinol
Tyrosinol
vaIin01
Isoleucinol
Methioninol
Leucinol
Serinol
AIaninol
INDUCTION Concentration (g/ml) 25 30 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10 500 100 50 10
1 OF TYPE C VIRUS Fzz
276 245 476 35 4 0 401 564 471 25 21 3 0 0 0 2 1 0 39 1 0 0 0 0 0 0 4 3 0 0 1, 0, 0, 0,
gh?-
(122)
[3H]I+ucme mcorporation* (%)
(239)
3.0 N.D. 6.0
(435)
3.5
5.0
160.0
21.0
70.0
90.0
3 0 0 0
49.0
“A total of 5 x Id induced cells was plated into NRK monolayers seeded 24 hr earlier in medium containing 2 pg/mI of polybrene at 1 x Id cells per 6cm dish. Induction period was 5 hr, except for 3-hr treatments given in parentheses. b C3H]Leucine (2 pCi/mI), L-leucine [4,5-3H(N)], New England Nuclear, 5 Ci/mmol) incorporation (6) w&s measured during the last 60 min of a 6-hr treatment with 500 ag of each alcohol. Incorporation was aIso measured in a noninduced control. All values were determined in triplicate.
195
nol, and valinol were the most effective inhibitors of protein synthesis in this cell system, each reducing [3H]leucine incorporation more than 95% following a 5-hr treatment. Methioninol reduced incorporation by 80%, and the other alcohols produced marginal effects. Concentrations of 0.25 mM tyrosinol and 0.50 mM histidinol gave near maximum induction during a 5-hr induction period. With tyrosinol, the most effective inducer, as little as a 3-hr treatment gave near maximum activation. Treatment of cells with each alcohol for longer periods did not appreciably change the levels of induction. To determine if induction frequencies could be increased above that found for each alcohol separately, histidinol and tyrosinol were added to cultures simultaneously for 5 or 16 hr at concentrations that gave either maximum (500 pg/ml) or submaximal activation. Under these conditions, the levels of induction did not increase. Viability of cells treated with either analog remained high during a 16-hr treatment. The addition of L-tyrosine at 20 pg/ml and r,-histidine at 50 pg/ml reduced the activation by the corresponding alcohol more than 90% proving these to be functional analogs in this cell system. Treatment of K-Balb cells with amino acid alcohols did not induce virus capable of forming foci on Balb/c or NIH swiss monolayers. The near maximum virus induction obtained within 3 hr after addition of tyrosinol suggested a rapid inhibition of protein synthesis. Incorporation of [3H]leucine was measured during 30-min intervals after addition of each alcohol at 0.5, 2, 4, and 6 hr, and induction of virus was followed at 1, 2, 3, 4, and 6 hr by infectious center assay (Fig. 1). During the first 30 min of treatment [3H]leucine incorporation was reduced 95% by tyrosinol and 80% by histidinol. Following a 2-hr treatment with tyrosin01 30-40s of maximum virus activation occurred. Histidinol and cycloheximide produced somewhat lower levels, but by 4 hr of treatment, all three chemicals had elicited 70-80s of their maximum induction capacity. Decay of virus expression following induction with either histidinol and tyrosin01 was rapid. Cells were induced with either analog (500 pg/ml) for 16 hr, and at
196
SHORT
COMMUNICATIONS
FIG. 1. Relationship between protein synthesis and virus induction. Cultures were treated with cycloheximide (25 pg/mI), histidinol (596 ag/mI), or tyrosinol (566 ag/mI) for variable time periods, and C3H]leucine incorporation and virus activation were measured concomitantly in duplicate dishes at the indicated times. Following treatment with inducers, cultures were treated with mitomycin C and seeded onto NRK monolayers. The data are expressed as percentages of the maximum induction obtained with each chemical following a 6-hr treatment. Incorporation with histidin01 (O), tyrosinol or cycloheximide (A). Virus induction with tyrosinol (A), histidinol (O), or cycloheximide (0).
tion times. This may reflect an accumulation of viral RNA with longer induction times (4). Histidinol and tyrosinol very efficiently activate type C virus from K-Balb cells in a readily reversible fashion following replacement with corresponding amino acids. Since virus induction following treatment with chemicals is a short-term process, the regulatory mechanism must turn over rapidly. The observation that valinol effrciently blocks protein synthesis without appreciable activation of virus may indicate a secondary process necessary for induction other than simply inhibition of protein synthesis. Chemicals of this nature should aid in the elucidation of molecular events necessary for virus activation. ACKNOWLEDGMENTS We are indebted to Dr. Marco Rabinovitz for helpful discussions and Dr. R. V. Gilden for continued interest and support. This work was supported by the Virus Cancer Program, Contract No. NOl-CO-75380, National Cancer Institute, NIH, Bethesda, Maryland 20614. REFERENCES
intervals following removal of inducer, cells were treated with mitomycin C and plated onto NRK monolayers for focus formation. By 20 hr following removal of either analog the number of foci had decreased to less than 5% of control values found at the time of removal of inducer. Twenty-four hours following removal of histidinol, [3H]leucine incorporation returned to normal levels. Activation of virus in cultures treated for 3 hr with histidinol or tyrosinol(500 pg/ml) was completely inhibited when either actinomycin D (1.2 pg/ml) or cytosine arabinoside (10 pg/ml) was present showing a requirement for RNA and DNA synthesis during induction. Cultures induced with histidinol for 2,3, or 4 hr followed by treatment with actinomycin D or cytosine arabinoside for 3 hr showed a decreasing sensitivity to posttreatment with longer induc-
1. LOWY, D. R., ROWE, W. P., TEICH, 2. 3. 4.
5. 6. 7’.
8. 9.
10.
N., and HARTLEY, J., Science 174, 155-156 (1971). AARONSON, S. A., TODARO, G. J., and SCOLNICK, E., Science 174, 157-159 (1971). AARONSON, S. A., and DUNN, C., Science 183, 422-424 (1974). CABRADILLA, C. D., ROBBINS, K. C., and AARONSON, S. A., Proc. Nat. Acad. Sci. USA 73, 4541-4545 (1976). AKSAMIT, R., and LONG, C. W., Virology 78, 567-570 (1977). AXSAMIT, R. R., CHRISTENSEN, W. L., and LONG, C. W., Virology 83,138-149 (1977). VAUGHAN, M. H., PAWLOWSKI, P. J., and FORCHAANMER, J., Proc. Nat. Acad. Sci. USA 68, 2057-2961 (1971). WARRINGTON, R. C., WRATTEN, N., and HECHTMAN, R., J. Biol. Chem. 262, 5251-5257 (1977). VAUGHAN, M. H., and HANSEN, B. S., J. Biol. Chem. 248.7087-7096 (1973). DUNN, C. Y., AARONSON, S. A., and STEPHENSON, J. R., Virology 66.579-588 (1975).