Volume 6 Number 4 April 1979
Nucleic Acids Research
Effect of toyocamycin on the synthesis of the 70S RNA of a murine retrovirus
C.J.Larsen, M.Mauchauff6, R.Hamelin, L.Peraudeau, L.Fedele and A.Tavitian
Laboratoire de Pharmacologie Exp6rimentale, Departement d'Oncologie Expeimentale (U. 107L.O.I.), Institut de Recherches sur les Maladies du Sang, Hopital St. Louis, 75475, Paris Cedex 10, France Received 15 February 1979 ABSTRACT
The murine Eveline cell line chronically infected by Friend virus was treated with Toyocamycin (TMC), an adenosin analog and the virions released in the presence of the drug were examined for their RNA. It was found that 70S RNA which was synthesized incorporated Toyocamycin. However, its subunit structure and its poly (A) content were apparently preserved. This incorporation may explain loss of endogenous reverse transcriptase activity.
INTRODUCTION
Toyocamycin (4 amino-5-cyano-7B-D-ribofuranosyl-pyrrolo 2-3-d pyrimidine) is an adenosine analog, which at low doses selectively incorporates in the 45S ribosomal RNA precursor and prevents its cleavage into 28S and 18S RNA (1). It has been shown that the drug does not prevent transcription of other cellular RNA species, since polysomal RNA having properties of messenger RNA is synthesized in the presence of TMC (2). The action of the drug on the replication of retroviruses was also studied and it was found that biosynthesis of virus was depressed by the same concentration range as that required for inhibition of ribosomal RNA synthesis (3-4). We have recently observed that virions produced in the course of the TMC treatment lost in-
fectivity and had a net decrease in their endogenous reverse transcriptase activity (5). Since this effect might result from an alteration of the viral genome, the 70S RNA of the TMC-treated virions was analyzed. In this study, we looked for the presence of TMC in the viral 70S RNA and showed that polyadenylation of the subunits occured in the presence of the drug.
MATERIALS AND METHODS
Cells The Eveline-Friend cells (6) were grown in suspension in Eagle's
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1547
Nucleic Acids Research minimum essential Medium (Flow) supplemented with 10 % heat inactivated fetal calf serum and without antibiotics. Conditions for labeling the cells have been reported elsewhere (7). with [3 P] (carrier free) or
[3H]3Adenosine
Virus purification
Virus was concentrated from the cell culture supernatants by Polyethylene glycol-6000 (Backer) which was added at a 8 % (w/v) final concentration. The suspensions were stirred for 2 h at 40C, then centrifuged at 10,000 rpm for 20 min. and dissolved in Tris-HCl 0,01M,pH 7.4, NaCl 0.1M, EDTA 0.001M (NTE, 1/10th to 1/20th of the initial volume). The resulting
turbid solution was layered on a 4 ml 20 % glycerol cushion (in NTE buffer) in a Beckman SW-27 rotor tube and centrifuged for 2 hours at 25 000 rpm. Finally, the pellet was redissolved in NTE (lml) and centrifuged at equilibrium in a 10-70 % sucrose gradient for 17 hours at 30 000 rpm (SW-41 Beckman rotor).
RNA isolation and fractionation
Viral RNA was extracted by sodium dodecylsulfate-phenol treatment as previously described (7). Poly (A) content was estimated by chromatography on oligo-dT-cellulose (7). Gel electrophoresis in polyacrylamide-agarose gels was performed according to Tiollais et al. (8).
Base analysis RNA was hydrolyzed by 0.5N KOH for 16h-18h at 370C. 2', 3' mononucleotides were separated by electrophoresis on Whatman 3M paper at pH 3.5,
0.5 % v/v Pyridine, 5 % v/v Acetic Acid). After drying, paper sheets were exposed against Kodirex X-ray films in order to locate the spots. For estimating relative proportions, spots were cut, immersed in scintillation liquid and counted in a Tricarb 3390.
Reagents
Toyocamycin (TMC) concentration was determined by spectrophotometric measurements. (Molar absorption at 279 nm, pH = 8.0 ; 1 mmole = 13.0).[ H] adenosine and[32p] were from the Commissariat a l'Energie Atomique - Saclay (France). was prepared by catalytic exchange of cold TMC in the "Laboratoire des molecules marquees - Commissariat a 1'Energie Atomique, Saclay". It was purified by paper chromatography in a Butanol-Methanol-Water Solvent. Specific activity of the product was 10 Ci/mmole. If necessary, it was diluted with cold TMC. Reverse transcriptase activity measurements Conditions were previously reported (9). Concentrations of viral
[3;Toyocamycin
1548
Nucleic Acids Research proteins were determined by a colorimetric method using Amido black dye (10). Equal amounts of virus
prepared according to fied virus
was
were
a
used in comparative tests. Viral
cores were
procedure precedently described (11). Briefly, puri-
incubated with
ionic detergent Sterox SL (Monsanto
a non
Company) and fractionated by equilibrium sucrose gradient.
RESULTS In
a
previous study,
we
have determined the concentration
TMC which stop the growth of Friend-Eveline cells (5). At 0.2 inhibits cellular growth and shuts off most of the ribosomal
ranges
jig/ml,
of
TMC
RNA processing.
Moreover, cell viability is maintained for at least 30 hours, while cytotoxicity is greatly enhanced when doses higher than 0.3
production is diminished and this effect is
eg/ml
are
used. Virus
pronounced when the dose of
more
TMC is increased. In order to evaluate the action of the drug
on
the viral RNA packa-
ged into virions, three identical cultures received 0.2 1
eg/ml
of TMC respectively. Fifty minutes later, 5
and after 16 hours labeling, virus
content. Presence of 70S RNA crose
was
was
0.5
eg/ml, one
2
were
a
0.2
eg/ml
concentration
was no more
fg/ml
was
was
su-
the 70S
located into low
trace of 70S RNA. This effect
inside the cells,
TMC concentrations higher than 0.2
added
purified and analyzed for its RNA
all the radioactivity
molecular weight RNA, whereas there is to be compared with the
e.C/ml
of
detected by sedimentation in velocity
gradient (not shown). Only at
RNA still present. At
0.5 pg and
tg,
as we
have precedently that
rapidly kill the cells (5).
Toyocamycin has been reported to incorporate into RNA molecules (5)To detect it in the 70S viral RNA, cells were exposed to[
H]Toyocamycin
for
16 hours. Virus was purified by banding in a sucrose gradient. A peak of
radioactivity located at the expected density of virus (1.15-1.16) was observed. Material of this peak was treated with
analyzed in
a sucrose
0.5
% sodium dodecylsulfate and
velocity gradient. A peak of RNA 70S was clearly resol-
ved (not shown). To confirm that tritium occurence in the 70S RNA did not
p2,] or
[4-.rMC labeled 70S RNAs were
result from
a
reutilization of [H]atoms,
digested by
a
0.5N KOH for 16 hours at 370C and the resulting mononucleotides
were
fractionated by paper electrophoresis at pH
3.5
(fig. 1). Besides the
spots corresponding to Cp,Ap,Gp,Up (from the origin to the top), two additional ones quoted with an arrow occured on the X-ray film. This is the
expected position for TMC monophosphate
and a degradation product formed
1549
Nucleic Acids Research
_
+
so +
I
3H cpm
TUC 1 Cp
Ap
TMC 2
Gp
Up
12%
2.113 22 %
is3
14% 2%
Analysis of nucleotides of TMC 70S RNA. RNA was labeled with 132] in presence of 0.2 1g/ml TMC for 16 hours or with the same dose of - TMC. 70S RNA was purified and hydrolyzed with KOH and the resulting p-, cleotides were separated on 3MM paper by high voltage electrophoresis. P] labeled spots were vizualised by exposing the dried paper against X-ray film. Counting of the 3H]TMC was done by cutting strips of paper and immersing them in scintillation fluid (represented by the diagram).
Figure 1
:
V FC/ml) [t
under alkaline conditions (12). Moreover, most of the
PH]radioactivity
was
located at the position of these 2 spots. This is a fair indication that the drug was incorporated into the RNA. An important part of (22 %) was also found at the front position of the spot corresponding to Cp. Identification of this spot is presently investigated.
[Hjradioactivity
Poly (A) content of TMC viral RNA was estimated by measuring retention of the 70S molecules on an oligo-dT-cellulose column. As seen in table I the relative proportions of RNA varied in each
experiment, but there
was
no difference between control and TMC-70S RNA. Moreover, it was observed that part of the TMC 70S RNA did not attach to the oligo-dT-cellulose if the
flow through the column was too rapid. This result implies that a Poly (A) segment is present in the TMC 70S RNA. To determine whether some of its A residues were substituted by TMC, 70S RNA was digested with pancreatic RNase. Conditions were chosen,
1550
Nucleic Acids Research
~~(-) Poly A.(+).RNA RNA Poly A~~~
. ,.
Rapid flow
Low flow
70S control RNA
63 %
37 %
70S TI RNA
69 %
31 %
70S control RNA
20 %
80 %
4%
96 %
70S TC RNA
Table I Poly (A) content of 70S RNA in TIC and control virions. STU cells were grown for 16 hours in the presence of 0.2 eg TMC. 32p (15 eC/ml) or 3H -Adenosine (10 PC/ml) were added 50 mn after the addition of the drug. Virus were purified and RNA was purified by Phenol-SDS extractions. 70S RNA was isolated from the bulk by velocity sucrose gradient and its ability to bind to oligo-dT-cellulose was tested. Rapid flow : sample loaded on the column and the column immediately washed with buffer. Low flow : sample loaded, allowed to remain on the column for 10 minutes before washing through with loading buffer.
[3+adenosine
celwhich did not destroy Poly (A) segment of the fraction of lular RNA retained on oligo-dT-cellulose. The Poly (A) nature of this material was asserted by testing its capacity to bind to nitrocellulose membrane at high ionic strength. In the experiment presented in table II, almst 8 % of the radioactivity of the TMC-70S RNA input was bound to a nitrocellulose
Cellular Poly (A) RNA + RNase
(Pancr.)
TMC - 70S viral RNA + RNase
High salt buffer
Low salt buffer
56 280 cpu (100 %)
-
7 670 cpm (13.6 %)
466 cpm (0.8 %)
29 500 cpm (100 %) 2 275 cpm (7.7 %)
-
248
cpu (0.8 %)
[H]-Toyocamycin
and binding Table II: Binding of TMC 70S RNA labeled with of [H|-Adenosine Poly (A) cellular RNA to nitrocellulose filters after treatment with RNase A. 70S viral RNA was labeled by growth of STU cells in medium contaiLig/ml [3H]-Trw for 16 hours and was separated from the bulk RNA by sucrose density gradient. 3 Cellular RNA was extracted from STU cells labelled for 3 hours with [ H]-Adenosine (20 eC/ml). Poly (A) RNA was isolated by chromatography on an oligo-dT-cellulose column. Appropriate amounts of each RNA were dissolved in 100 pl of digestion buffer 0.01M, pH 7.4 Tris-HCl, 0.3M NaCl, 0.001M EDTA, 20 eg/ml soluble yeast RNA and treated with 2 tg/ml of Pancreatic RNase (Sigma) for 30 mn at 370C. The solutions were diluted in 4 ml of high salt Buffer (0.01M Tris-HCl pH 7.5, 0.5M NaCl, 0.01M Mg Cl2) or 4 ml of low salt buffer (0.05M NaCl, 0.0114 Tris-HCl) and slowly filtered on 0.22 e nitrocellulose membranes presoaked in the buffer.
ning 0.2
1551
Nucleic Acids Research filter after pancreatic RNase digestion. More than 90 % of this material was Poly (A), since it was not fixed onto the membrane, when Sodium Chloride molarity of the buffer was decreased from 0.5 to 0.05M. To estimate the size labelled 70S RNA was prepared and digested with of the Poly A tails, pancreatic RNase. The poly (A) containing material was separated from the bulk of radioactivity by filtration on nitrocellulose membrane at 0.5M NaCl
[3+4TMC
concentration. After elution, this material was analyzed in a 10 % polyacrylamide gel. Most of the radioactivity moved at the position of two bands which by comparison with 4S and 5S RNA were estimated to have a 25 and 40 nucleotides lenght (not shown). The 70S RNA of the retrovirus is a specific agregate of two 30-35S identical subunits (13). In order to see if this structure was conserved in the 70S RNA of virions synthesized in the presence of TMC, two identical cell cultures were prepared. One of them received 0.2 tg/ml TMC and 50 min. later, 1 mc 3H]Adenosine was added to the two spinners. After 15 hours, virus was harvested and purified. Its 70S RNA was isolated by sucrose gradient centrifugation and heated at 800 for one minut in order to abolish its secondary structure. This operation was followed by an oligo-dT-column step, so that only Poly (A)-containing molecules were conserved for polyacrylamide gel analysis (fig. 2). It can be seen that the heated RNA of the control virus consisted of an homogeneous peak moving in the 35S region of the gel (fig. 2, o-*). The RNA of the TMC virions was more heterogeneous but an appreciable amount (40 %) co-migrated with the 35S subunit suggesting that a least a part of the viral genome was still in this structure. The same result was obtained where labelling of the cells was performed with [34+TMC instead of [+Adenosine. This indicates that TMC was also incorporated in the 35S
subunit. Since Toyocamycin is incorporated into the 70S RNA during the course of its biosynthesis, it was interesting to investigate its possible effects on the endogenous reverse transcriptase activity of the TMC virions. In a previous work, we reported a decreased activity of the enzyme using its natural template, when identical amounts of control and TMC-treated virions used in place were employed (5). This result was also obtained as a DNA precursor. To confirm it, we prepared viral cores from of
with[3HIdGTP
PHITTP
control and TMC virions by Sterox SL treatment followed by sucrose gradient (11). The material running in the 1.22-1.25 g/cc density region of the gradients was pooled and assayed for endogenous reverse transcriptase activity. From the data presented in figure 3, it is evident that no enzymatic activity is present in the cores extracted from the TMC-virions. However, the same 1552
Nucleic Acids Research 70S
c3Hml cpm
Figure 2 : Gel electrophoresis of RNA extracted from control and TMC-treated virions. The RNA was extracted from purified virus preparations and chromatographied on an oligo-dT-cellulose column. Poly (A) containing RNA was precipitated by ethanol, dissolved in NTE buffer and heated at 800C for one minute. The samples were loaded onto 1.7 % polyacrylamide - 0.5 % agarose composite gels. 1.5 mm slices were cut, hydrolyzed in 0.5 ml H 0 at 370C overnight and counted with 10 ml Soluene (Packard). (- : 78S RNA from o TMC virions; o-o : heated RNA from control virus; heated RNA from TMC virions).
[+TTP
preparations gave identical incorporations of in an exogenous reaction using Poly rA-oligodT as template (56780 cpm and 57280 cpm), suggesting that the reverse transcriptase was not impaired by TMC.
DISCUSSION
In a previous work (5), we have reported that Toyocamycin decreases
the production and the infectivity of retroviruses grown in culture. Since the drug is known to be incorporated into the RNA and interferes with the processing of 45S ribosomal precursor, we though that a similar mechanism could occur during the synthesis of the viral RNA in the TMC-treated cells. Our data show that the viral RNA was present in the 70S form when the TMC concentration did not exceed 0.2 eg/ml. Its subunit structure and its poly (A) content were conserved. Results of experiments performed with
1553
Nucleic Acids Research
3H cpm 310 CONTROL
210'
10./ TMC
30
90
TIME
Figure 3 Kinetics of incorporation of [+TTP into DNA in the presence of viral cores. Equal amounts of viral proteins were added to the reaction mixtures. Aliquots were TCA precipitated at the indicated times and filtered on nitrocellulose membranes. tritiated Toyocamycin and analysis of the mononucleotides after alkaline hydrolysis of RNA, showed that the analog was incorporated in the RNA. The extent of this incorporation was dependent on the dose of drug added to the
cell culture (umpublished results). In experiments using [3 2P]labeling, it was found that 2 % of the radioactivity was located in the TMC spot and its
by-product. From this data we can assume that 4 to 6 % of Adenosine is replaced by the antibiotic in the viral RNA, when it is used at a 0.2 Ug/ml concentration. The main consequence of such an incorporation should be a structural change in the secondary structure. This alterations might decrease the stability of duplex structures between nucleic acids. In experiments using cellular RNA extracted from TMC-treated STU cells, we have observed that a significant amount of viral sequences are apparently lost, as judged by the percentage of complementary viral DNA protected by a saturating amount of RNA. This, in fact, results from a higher sensitivity of the hybrids to the S1 nuclease since this phenomenon does not occur when hybrids are analyzed on hydroxyapatite columns. This sensitivity is readily explained by the existence of local single stranded segments near sites of TMC integration, allowing the S1 nuclease to work.
1554
Nucleic Acids Research Another consequence of the Toyocamycin incorporation could possibly result in a miscoded message, whose translational capacity can be tested in an in vitro cell-free synthesis system. However, this possibility remains to be demonstrated.
Other authors have recently published results suggesting that TMC prevents polyadenylation of the heterogenous nuclear RNA during late lytic infection of HeLa S3 cells by adenovirus (13). This is not in agreement with our results, since we found that viral 70S RNA was polyadenylated in the presence of the analog. However the dose of TMC used in the experiments and the duration of the exposure of the cells to the drug were completely different,
since a 10 times higher dose and a 16 times shorter period of time were used by these authors. We have mentioned precedently that 70S RNA does not occur in viral particles, when a concentration higher than 0.5 eg/ml is used. It is conceivable that cytotoxicity of TMC activates molecular mechanisms which facilitate RNA degradation and destroy Poly (A) segment or specific sites necessary to the initiation of their synthesis. Alternatively, the drug inhibits Poly (A) synthesis at doses higher than the one we used. In fact, such mechanisms can occur under our experimental conditions since we have observed that the size of the Poly (A) segment is greatly reduced by comparison with that of the control, whose average lenght has been estimated to 100-200 nucleotides (14). We have already reported that the endogenous reverse transcriptase activity of the TMC-treated virions was diminished, although exogenous activity using Poly rA-oligodT or Poly rC-oligodG was entirely similar wether the enzyme consisted of normal virions or TMC-treated virions (5). This result suggests that the enzyme is not modified but cannot transcribe correctly the viral genome whose structure is impaired by the presence of TMC. In that case, it would be interesting to determine if the drug is preferentially incorporated at some specific sites of the viral subunits.
ACKNOWLEDGMENTS This investigation was supported by a grant from the Institut National de la Sante et de la Recherche Medicale - A.T.P. NO 52-77-84.
REFERENCES 1 - Tavitian, A., 157, 33-42.
Uretsky, S.C. and Acs, G. (1968) Biochim. Biophys. Acta
1555
Nucleic Acids Research 2 - Tavitian, A., Uretsky, S.C. and Acs, G. (1969) Biochim. Biophys. Acta
179, 50-57. 3 - Bonar, R.A., Chabot, J.F., Langlois, A.J., Sverak, L., Veprek, L. and Beard, J.W. (1970) Cancer Research 30, 753-762. 4 - Riman, J. (1971) Lepetit colloquia on Biology and Medicine. The biology of oncogenic viruses (Silvestri Edit. North.) 232-236. 5 - Mauchauffe, M., Hamelin, R., Michel, M.L. and Larsen, C.J. (1979) Biomedicine (to be published). 6 - Moennig, V., Frank, H., Hunsmann, G., Schneider, I. and Schafer, W. (1974) Virology 61, 100-111. 7 - Michel, M.L., Roussel, M., Poitevin, E., Samso, A. and Larsen, C.J.
(1977). 8 - Tiollais, P., Galibert, F., Lepetit, A. and Auger, M.A. (1972) Biochimie 54, 339-354. 9 - Tavitian, A., Hamelin, R., Tchen, P., Olofsson, B. and Boiron, M. (1974) Proc. Nat. Ac. Sci. USA 71, 755-759. 10- Kuno, H. and Kihara, H.K. (1967) Nature 215, 974-976. 11- Larsen, C.J., Emanoil-Ravicovitch, R., Samso, A., Robin, J., Tavitian, A. and Boiron, M. (1973) Virology 54, 552-556. 12- Swart, C. and Hodge, L.D. (1978) Virology 84, 374-389. 13- Kung, H.J., Barley, J.M., Davidson, N., Nicolson, M.O. and McAllister, R. (1975) J. Virol. 16, 397-411. 14- Lai, M.C. and Duesberg, P.H. (1972) Nature, 235, 383-386.
1556
Nucleic Acids Research
Effect of toyocamycin on the synthesis of the 70S RNA of a murine retrovirus
C.J.Larsen, M.Mauchauff6, R.Hamelin, L.Peraudeau, L.Fedele and A.Tavitian
Laboratoire de Pharmacologie Exp6rimentale, Departement d'Oncologie Expeimentale (U. 107L.O.I.), Institut de Recherches sur les Maladies du Sang, Hopital St. Louis, 75475, Paris Cedex 10, France Received 15 February 1979 ABSTRACT
The murine Eveline cell line chronically infected by Friend virus was treated with Toyocamycin (TMC), an adenosin analog and the virions released in the presence of the drug were examined for their RNA. It was found that 70S RNA which was synthesized incorporated Toyocamycin. However, its subunit structure and its poly (A) content were apparently preserved. This incorporation may explain loss of endogenous reverse transcriptase activity.
INTRODUCTION
Toyocamycin (4 amino-5-cyano-7B-D-ribofuranosyl-pyrrolo 2-3-d pyrimidine) is an adenosine analog, which at low doses selectively incorporates in the 45S ribosomal RNA precursor and prevents its cleavage into 28S and 18S RNA (1). It has been shown that the drug does not prevent transcription of other cellular RNA species, since polysomal RNA having properties of messenger RNA is synthesized in the presence of TMC (2). The action of the drug on the replication of retroviruses was also studied and it was found that biosynthesis of virus was depressed by the same concentration range as that required for inhibition of ribosomal RNA synthesis (3-4). We have recently observed that virions produced in the course of the TMC treatment lost in-
fectivity and had a net decrease in their endogenous reverse transcriptase activity (5). Since this effect might result from an alteration of the viral genome, the 70S RNA of the TMC-treated virions was analyzed. In this study, we looked for the presence of TMC in the viral 70S RNA and showed that polyadenylation of the subunits occured in the presence of the drug.
MATERIALS AND METHODS
Cells The Eveline-Friend cells (6) were grown in suspension in Eagle's
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1547
Nucleic Acids Research minimum essential Medium (Flow) supplemented with 10 % heat inactivated fetal calf serum and without antibiotics. Conditions for labeling the cells have been reported elsewhere (7). with [3 P] (carrier free) or
[3H]3Adenosine
Virus purification
Virus was concentrated from the cell culture supernatants by Polyethylene glycol-6000 (Backer) which was added at a 8 % (w/v) final concentration. The suspensions were stirred for 2 h at 40C, then centrifuged at 10,000 rpm for 20 min. and dissolved in Tris-HCl 0,01M,pH 7.4, NaCl 0.1M, EDTA 0.001M (NTE, 1/10th to 1/20th of the initial volume). The resulting
turbid solution was layered on a 4 ml 20 % glycerol cushion (in NTE buffer) in a Beckman SW-27 rotor tube and centrifuged for 2 hours at 25 000 rpm. Finally, the pellet was redissolved in NTE (lml) and centrifuged at equilibrium in a 10-70 % sucrose gradient for 17 hours at 30 000 rpm (SW-41 Beckman rotor).
RNA isolation and fractionation
Viral RNA was extracted by sodium dodecylsulfate-phenol treatment as previously described (7). Poly (A) content was estimated by chromatography on oligo-dT-cellulose (7). Gel electrophoresis in polyacrylamide-agarose gels was performed according to Tiollais et al. (8).
Base analysis RNA was hydrolyzed by 0.5N KOH for 16h-18h at 370C. 2', 3' mononucleotides were separated by electrophoresis on Whatman 3M paper at pH 3.5,
0.5 % v/v Pyridine, 5 % v/v Acetic Acid). After drying, paper sheets were exposed against Kodirex X-ray films in order to locate the spots. For estimating relative proportions, spots were cut, immersed in scintillation liquid and counted in a Tricarb 3390.
Reagents
Toyocamycin (TMC) concentration was determined by spectrophotometric measurements. (Molar absorption at 279 nm, pH = 8.0 ; 1 mmole = 13.0).[ H] adenosine and[32p] were from the Commissariat a l'Energie Atomique - Saclay (France). was prepared by catalytic exchange of cold TMC in the "Laboratoire des molecules marquees - Commissariat a 1'Energie Atomique, Saclay". It was purified by paper chromatography in a Butanol-Methanol-Water Solvent. Specific activity of the product was 10 Ci/mmole. If necessary, it was diluted with cold TMC. Reverse transcriptase activity measurements Conditions were previously reported (9). Concentrations of viral
[3;Toyocamycin
1548
Nucleic Acids Research proteins were determined by a colorimetric method using Amido black dye (10). Equal amounts of virus
prepared according to fied virus
was
were
a
used in comparative tests. Viral
cores were
procedure precedently described (11). Briefly, puri-
incubated with
ionic detergent Sterox SL (Monsanto
a non
Company) and fractionated by equilibrium sucrose gradient.
RESULTS In
a
previous study,
we
have determined the concentration
TMC which stop the growth of Friend-Eveline cells (5). At 0.2 inhibits cellular growth and shuts off most of the ribosomal
ranges
jig/ml,
of
TMC
RNA processing.
Moreover, cell viability is maintained for at least 30 hours, while cytotoxicity is greatly enhanced when doses higher than 0.3
production is diminished and this effect is
eg/ml
are
used. Virus
pronounced when the dose of
more
TMC is increased. In order to evaluate the action of the drug
on
the viral RNA packa-
ged into virions, three identical cultures received 0.2 1
eg/ml
of TMC respectively. Fifty minutes later, 5
and after 16 hours labeling, virus
content. Presence of 70S RNA crose
was
was
0.5
eg/ml, one
2
were
a
0.2
eg/ml
concentration
was no more
fg/ml
was
was
su-
the 70S
located into low
trace of 70S RNA. This effect
inside the cells,
TMC concentrations higher than 0.2
added
purified and analyzed for its RNA
all the radioactivity
molecular weight RNA, whereas there is to be compared with the
e.C/ml
of
detected by sedimentation in velocity
gradient (not shown). Only at
RNA still present. At
0.5 pg and
tg,
as we
have precedently that
rapidly kill the cells (5).
Toyocamycin has been reported to incorporate into RNA molecules (5)To detect it in the 70S viral RNA, cells were exposed to[
H]Toyocamycin
for
16 hours. Virus was purified by banding in a sucrose gradient. A peak of
radioactivity located at the expected density of virus (1.15-1.16) was observed. Material of this peak was treated with
analyzed in
a sucrose
0.5
% sodium dodecylsulfate and
velocity gradient. A peak of RNA 70S was clearly resol-
ved (not shown). To confirm that tritium occurence in the 70S RNA did not
p2,] or
[4-.rMC labeled 70S RNAs were
result from
a
reutilization of [H]atoms,
digested by
a
0.5N KOH for 16 hours at 370C and the resulting mononucleotides
were
fractionated by paper electrophoresis at pH
3.5
(fig. 1). Besides the
spots corresponding to Cp,Ap,Gp,Up (from the origin to the top), two additional ones quoted with an arrow occured on the X-ray film. This is the
expected position for TMC monophosphate
and a degradation product formed
1549
Nucleic Acids Research
_
+
so +
I
3H cpm
TUC 1 Cp
Ap
TMC 2
Gp
Up
12%
2.113 22 %
is3
14% 2%
Analysis of nucleotides of TMC 70S RNA. RNA was labeled with 132] in presence of 0.2 1g/ml TMC for 16 hours or with the same dose of - TMC. 70S RNA was purified and hydrolyzed with KOH and the resulting p-, cleotides were separated on 3MM paper by high voltage electrophoresis. P] labeled spots were vizualised by exposing the dried paper against X-ray film. Counting of the 3H]TMC was done by cutting strips of paper and immersing them in scintillation fluid (represented by the diagram).
Figure 1
:
V FC/ml) [t
under alkaline conditions (12). Moreover, most of the
PH]radioactivity
was
located at the position of these 2 spots. This is a fair indication that the drug was incorporated into the RNA. An important part of (22 %) was also found at the front position of the spot corresponding to Cp. Identification of this spot is presently investigated.
[Hjradioactivity
Poly (A) content of TMC viral RNA was estimated by measuring retention of the 70S molecules on an oligo-dT-cellulose column. As seen in table I the relative proportions of RNA varied in each
experiment, but there
was
no difference between control and TMC-70S RNA. Moreover, it was observed that part of the TMC 70S RNA did not attach to the oligo-dT-cellulose if the
flow through the column was too rapid. This result implies that a Poly (A) segment is present in the TMC 70S RNA. To determine whether some of its A residues were substituted by TMC, 70S RNA was digested with pancreatic RNase. Conditions were chosen,
1550
Nucleic Acids Research
~~(-) Poly A.(+).RNA RNA Poly A~~~
. ,.
Rapid flow
Low flow
70S control RNA
63 %
37 %
70S TI RNA
69 %
31 %
70S control RNA
20 %
80 %
4%
96 %
70S TC RNA
Table I Poly (A) content of 70S RNA in TIC and control virions. STU cells were grown for 16 hours in the presence of 0.2 eg TMC. 32p (15 eC/ml) or 3H -Adenosine (10 PC/ml) were added 50 mn after the addition of the drug. Virus were purified and RNA was purified by Phenol-SDS extractions. 70S RNA was isolated from the bulk by velocity sucrose gradient and its ability to bind to oligo-dT-cellulose was tested. Rapid flow : sample loaded on the column and the column immediately washed with buffer. Low flow : sample loaded, allowed to remain on the column for 10 minutes before washing through with loading buffer.
[3+adenosine
celwhich did not destroy Poly (A) segment of the fraction of lular RNA retained on oligo-dT-cellulose. The Poly (A) nature of this material was asserted by testing its capacity to bind to nitrocellulose membrane at high ionic strength. In the experiment presented in table II, almst 8 % of the radioactivity of the TMC-70S RNA input was bound to a nitrocellulose
Cellular Poly (A) RNA + RNase
(Pancr.)
TMC - 70S viral RNA + RNase
High salt buffer
Low salt buffer
56 280 cpu (100 %)
-
7 670 cpm (13.6 %)
466 cpm (0.8 %)
29 500 cpm (100 %) 2 275 cpm (7.7 %)
-
248
cpu (0.8 %)
[H]-Toyocamycin
and binding Table II: Binding of TMC 70S RNA labeled with of [H|-Adenosine Poly (A) cellular RNA to nitrocellulose filters after treatment with RNase A. 70S viral RNA was labeled by growth of STU cells in medium contaiLig/ml [3H]-Trw for 16 hours and was separated from the bulk RNA by sucrose density gradient. 3 Cellular RNA was extracted from STU cells labelled for 3 hours with [ H]-Adenosine (20 eC/ml). Poly (A) RNA was isolated by chromatography on an oligo-dT-cellulose column. Appropriate amounts of each RNA were dissolved in 100 pl of digestion buffer 0.01M, pH 7.4 Tris-HCl, 0.3M NaCl, 0.001M EDTA, 20 eg/ml soluble yeast RNA and treated with 2 tg/ml of Pancreatic RNase (Sigma) for 30 mn at 370C. The solutions were diluted in 4 ml of high salt Buffer (0.01M Tris-HCl pH 7.5, 0.5M NaCl, 0.01M Mg Cl2) or 4 ml of low salt buffer (0.05M NaCl, 0.0114 Tris-HCl) and slowly filtered on 0.22 e nitrocellulose membranes presoaked in the buffer.
ning 0.2
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Nucleic Acids Research filter after pancreatic RNase digestion. More than 90 % of this material was Poly (A), since it was not fixed onto the membrane, when Sodium Chloride molarity of the buffer was decreased from 0.5 to 0.05M. To estimate the size labelled 70S RNA was prepared and digested with of the Poly A tails, pancreatic RNase. The poly (A) containing material was separated from the bulk of radioactivity by filtration on nitrocellulose membrane at 0.5M NaCl
[3+4TMC
concentration. After elution, this material was analyzed in a 10 % polyacrylamide gel. Most of the radioactivity moved at the position of two bands which by comparison with 4S and 5S RNA were estimated to have a 25 and 40 nucleotides lenght (not shown). The 70S RNA of the retrovirus is a specific agregate of two 30-35S identical subunits (13). In order to see if this structure was conserved in the 70S RNA of virions synthesized in the presence of TMC, two identical cell cultures were prepared. One of them received 0.2 tg/ml TMC and 50 min. later, 1 mc 3H]Adenosine was added to the two spinners. After 15 hours, virus was harvested and purified. Its 70S RNA was isolated by sucrose gradient centrifugation and heated at 800 for one minut in order to abolish its secondary structure. This operation was followed by an oligo-dT-column step, so that only Poly (A)-containing molecules were conserved for polyacrylamide gel analysis (fig. 2). It can be seen that the heated RNA of the control virus consisted of an homogeneous peak moving in the 35S region of the gel (fig. 2, o-*). The RNA of the TMC virions was more heterogeneous but an appreciable amount (40 %) co-migrated with the 35S subunit suggesting that a least a part of the viral genome was still in this structure. The same result was obtained where labelling of the cells was performed with [34+TMC instead of [+Adenosine. This indicates that TMC was also incorporated in the 35S
subunit. Since Toyocamycin is incorporated into the 70S RNA during the course of its biosynthesis, it was interesting to investigate its possible effects on the endogenous reverse transcriptase activity of the TMC virions. In a previous work, we reported a decreased activity of the enzyme using its natural template, when identical amounts of control and TMC-treated virions used in place were employed (5). This result was also obtained as a DNA precursor. To confirm it, we prepared viral cores from of
with[3HIdGTP
PHITTP
control and TMC virions by Sterox SL treatment followed by sucrose gradient (11). The material running in the 1.22-1.25 g/cc density region of the gradients was pooled and assayed for endogenous reverse transcriptase activity. From the data presented in figure 3, it is evident that no enzymatic activity is present in the cores extracted from the TMC-virions. However, the same 1552
Nucleic Acids Research 70S
c3Hml cpm
Figure 2 : Gel electrophoresis of RNA extracted from control and TMC-treated virions. The RNA was extracted from purified virus preparations and chromatographied on an oligo-dT-cellulose column. Poly (A) containing RNA was precipitated by ethanol, dissolved in NTE buffer and heated at 800C for one minute. The samples were loaded onto 1.7 % polyacrylamide - 0.5 % agarose composite gels. 1.5 mm slices were cut, hydrolyzed in 0.5 ml H 0 at 370C overnight and counted with 10 ml Soluene (Packard). (- : 78S RNA from o TMC virions; o-o : heated RNA from control virus; heated RNA from TMC virions).
[+TTP
preparations gave identical incorporations of in an exogenous reaction using Poly rA-oligodT as template (56780 cpm and 57280 cpm), suggesting that the reverse transcriptase was not impaired by TMC.
DISCUSSION
In a previous work (5), we have reported that Toyocamycin decreases
the production and the infectivity of retroviruses grown in culture. Since the drug is known to be incorporated into the RNA and interferes with the processing of 45S ribosomal precursor, we though that a similar mechanism could occur during the synthesis of the viral RNA in the TMC-treated cells. Our data show that the viral RNA was present in the 70S form when the TMC concentration did not exceed 0.2 eg/ml. Its subunit structure and its poly (A) content were conserved. Results of experiments performed with
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3H cpm 310 CONTROL
210'
10./ TMC
30
90
TIME
Figure 3 Kinetics of incorporation of [+TTP into DNA in the presence of viral cores. Equal amounts of viral proteins were added to the reaction mixtures. Aliquots were TCA precipitated at the indicated times and filtered on nitrocellulose membranes. tritiated Toyocamycin and analysis of the mononucleotides after alkaline hydrolysis of RNA, showed that the analog was incorporated in the RNA. The extent of this incorporation was dependent on the dose of drug added to the
cell culture (umpublished results). In experiments using [3 2P]labeling, it was found that 2 % of the radioactivity was located in the TMC spot and its
by-product. From this data we can assume that 4 to 6 % of Adenosine is replaced by the antibiotic in the viral RNA, when it is used at a 0.2 Ug/ml concentration. The main consequence of such an incorporation should be a structural change in the secondary structure. This alterations might decrease the stability of duplex structures between nucleic acids. In experiments using cellular RNA extracted from TMC-treated STU cells, we have observed that a significant amount of viral sequences are apparently lost, as judged by the percentage of complementary viral DNA protected by a saturating amount of RNA. This, in fact, results from a higher sensitivity of the hybrids to the S1 nuclease since this phenomenon does not occur when hybrids are analyzed on hydroxyapatite columns. This sensitivity is readily explained by the existence of local single stranded segments near sites of TMC integration, allowing the S1 nuclease to work.
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Nucleic Acids Research Another consequence of the Toyocamycin incorporation could possibly result in a miscoded message, whose translational capacity can be tested in an in vitro cell-free synthesis system. However, this possibility remains to be demonstrated.
Other authors have recently published results suggesting that TMC prevents polyadenylation of the heterogenous nuclear RNA during late lytic infection of HeLa S3 cells by adenovirus (13). This is not in agreement with our results, since we found that viral 70S RNA was polyadenylated in the presence of the analog. However the dose of TMC used in the experiments and the duration of the exposure of the cells to the drug were completely different,
since a 10 times higher dose and a 16 times shorter period of time were used by these authors. We have mentioned precedently that 70S RNA does not occur in viral particles, when a concentration higher than 0.5 eg/ml is used. It is conceivable that cytotoxicity of TMC activates molecular mechanisms which facilitate RNA degradation and destroy Poly (A) segment or specific sites necessary to the initiation of their synthesis. Alternatively, the drug inhibits Poly (A) synthesis at doses higher than the one we used. In fact, such mechanisms can occur under our experimental conditions since we have observed that the size of the Poly (A) segment is greatly reduced by comparison with that of the control, whose average lenght has been estimated to 100-200 nucleotides (14). We have already reported that the endogenous reverse transcriptase activity of the TMC-treated virions was diminished, although exogenous activity using Poly rA-oligodT or Poly rC-oligodG was entirely similar wether the enzyme consisted of normal virions or TMC-treated virions (5). This result suggests that the enzyme is not modified but cannot transcribe correctly the viral genome whose structure is impaired by the presence of TMC. In that case, it would be interesting to determine if the drug is preferentially incorporated at some specific sites of the viral subunits.
ACKNOWLEDGMENTS This investigation was supported by a grant from the Institut National de la Sante et de la Recherche Medicale - A.T.P. NO 52-77-84.
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Uretsky, S.C. and Acs, G. (1968) Biochim. Biophys. Acta
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179, 50-57. 3 - Bonar, R.A., Chabot, J.F., Langlois, A.J., Sverak, L., Veprek, L. and Beard, J.W. (1970) Cancer Research 30, 753-762. 4 - Riman, J. (1971) Lepetit colloquia on Biology and Medicine. The biology of oncogenic viruses (Silvestri Edit. North.) 232-236. 5 - Mauchauffe, M., Hamelin, R., Michel, M.L. and Larsen, C.J. (1979) Biomedicine (to be published). 6 - Moennig, V., Frank, H., Hunsmann, G., Schneider, I. and Schafer, W. (1974) Virology 61, 100-111. 7 - Michel, M.L., Roussel, M., Poitevin, E., Samso, A. and Larsen, C.J.
(1977). 8 - Tiollais, P., Galibert, F., Lepetit, A. and Auger, M.A. (1972) Biochimie 54, 339-354. 9 - Tavitian, A., Hamelin, R., Tchen, P., Olofsson, B. and Boiron, M. (1974) Proc. Nat. Ac. Sci. USA 71, 755-759. 10- Kuno, H. and Kihara, H.K. (1967) Nature 215, 974-976. 11- Larsen, C.J., Emanoil-Ravicovitch, R., Samso, A., Robin, J., Tavitian, A. and Boiron, M. (1973) Virology 54, 552-556. 12- Swart, C. and Hodge, L.D. (1978) Virology 84, 374-389. 13- Kung, H.J., Barley, J.M., Davidson, N., Nicolson, M.O. and McAllister, R. (1975) J. Virol. 16, 397-411. 14- Lai, M.C. and Duesberg, P.H. (1972) Nature, 235, 383-386.
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