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Size differences among the high molecular weight RNA's of avian tumor viruses
VIROLOGY
43, 214-222 (1971)
Size Differences
among
the High Molecular
of Avian DAN1 Max-Plan&-Znstitut
fiir
RNA’S
Tumor Viruses
P. BOLOGNESI’ Virusforschung,
Weight
AND
THOMAS
Biologisch-medizinische
GRAF Abteilung,
Tiibingen,
Germany
Accepted October 1, 1970 Sedimentation analyses in sucrose gradients of high molecular weight RNAs from several strains of avian tumor viruses revealed considerable variations. Analysis in gel electrophoresis likewise revealed differences in the rate of migration and experiments with various ionic conditions suggested that the observed differences are probably not due to conformational effects in the molecules but reflect variations in molecular size. The maximum difference obtained corresponds to a molecular weight of about 1.3 X lo6 daltons. The observed differences were not correlated to (i) whether the agent was a sarcoma or leukosis virus or (ii) the virus subgroup, or (iii) the capacity of the virus to form tumors in mammals. It was shown, however, that mutagenization of a sarcoma virus to an infectious nonconverting virus was accompanied by a loss in molecular size of the RNA. The significance of these results is discussed. INTRODUCTION
In studies with the high molecular weight (HMW) RNAs from certain strains of avian tumor viruses, we noticed slight differences in the sedimentation behavior of the molecules. That such differences in sedimentation rates had not been previously observed may have been due to the virus strains studied. The sedimentation behavior of the HMW RNAs from a mixture of Rous sarcoma virus (RSV) and Rous-associated virus (RAV) in comparison to avian myeloblastosis virus (AMV), the latter also being a mixed strain (Moscovici and Vogt, 1968) revealed no differences (Robinson and Baluda, 1965). In the work reported here strains of known classification were employed, which in the case of sarcoma viruses were purified by single focus cloning. However, AMV, which in our laboratory consists mainly of subgroup B virus was also used for some analyses. 1 Recipient of Public Health Service Postdoctoral Fellowship No. l-F2-CA-38, 592-01 from the National Cancer Institute.
Avian RNA tumor viruses are classified according to some of their general properties. They may, for practical purposes, be subdivided into sarcoma and leukosis viruses depending on their ability to convert chicken fibroblasts. Viruses of both types can be further subdivided into four groups as determined by their envelope properties (Vogt and Ishizaki, 1966; Duff and Vogt, 1969). It became of interest to determine whether the sedimentation properties of the RNAs could be correlated with any of these functions. Since previous work (Gold& 1970; Toyoshima et al., 1970; Graf et al., 1971) suggested that nonconverting viruses might originate from sarcoma viruses by mutation, it was of particular interest to determine whether the genomes of sarcoma viruses were larger than those of leukosis viruses. Although the studies revealed that the latter is not a general case, it could be demonstrated that mutagenization of a sarcoma virus gave rise to an infectious agent which had lost its converting ability and whose RNA was considerably smaller than that of the parent sarcoma strain. 214
AVIAN MATERIALS
TUMOR
AND METHODS
Buffers TSE: 0.01 M Tris. HCl, 0.1 M NaCl, 0.001 M EDTA, pH 7.2. Electrophoresis Buffer (E buffer): 0.04 M TriseHCl, 0.02 M Naf acetate, 0.001 M EDTA, 0.1% SDS, pH 7.4. Low Salt Buffer: 0.03 M Tris.HCl, 0.016 M HCI, 0.0001 M EDTA, 0.1 % SDS, pH 7.8. Low-salt, Mgz+ buffer: 0.03 M Tris .HCl, 0.016 M HCl 0.0001 M EDTA, 0.02 M Al@+ acetate, 0.1% SDS, pH 7.8. Viruses ancl cells. The origins of the Schmidt Ruppin strain 1 of Rous sarcoma virus (SRV-l), of SRV-H, nonconverting SRV-1 (NC-SRV-l), NC-SRV-H, and RAV-1 were described in a separate communication (Graf et al., 1971). A Schmidt Ruppin virus obtained from Dr. Alice Gold6 (SRV-G) was also employed (Graf et al., 1971). Rous-associated virus 50 (RAV-50) and RAV-7 were kindly supplied by Dr. P. Ii. Vogt, Seattle, Washington. The MC-29 strain was obtained through the courtesy of Dr. A. J. Langlois, and the MC-B was derived by focus cloning and belongs to subgroup B (unpublished results). The BAI-A strain of AYIV was originally obtained from Dr. J. W. Beard, Durham, North Carolina. AMV was isolated from the plasma of leukemic chickens and in our laboratory consists mainly of virus from subgroup B. The isolation and propagation of MAV-B was reported earlier (Bauer and Graf, 1969). All viruses were grown on susceptible chicken embryo cells which were selected according to the classification of the virus used (Graf et al., 1971). Growth of radioactive virus. Labeling of virus grown in chicken embryo fibroblast cultures with uridine was performed in a modified Eagle medium in the presence of 6% heat inactivated calf serum. Incubation with 20 ~Ci/ml uridine-3H (20 mCi/mmole, Radiochemical Centre, Amersham, England) was for 18 hr at 37”. Before incorporation of 32P, the cells were washed 3 times with phosphate-free Eagle medium. They were then incubated at 37” for 18 hr with 100 &X/ml 32P-orthophosphate (carrier free, Radiochemical Centre,
VIRUS
RNAs
215
Amersham, England) in phosphate-free medium containing 6 % dialyzed calf serum. Myeloblast cultures (Langlois et al., 1966> to be labeled with 32Pwere similarly washed, but 20% dialyzed calf serum was employed during labeling. Isolation of RNA. For these experiments extensive purification of the viruses was not found to be necessary. Therefore two cycles of alternate high and low speed centrifugation were employed to recover the virus. The final pellets were taken up in TSE. buffer, combined in appropriate proportions according to radioactivity, and extracted with phenol and SDS at 4”. The phenol used was saturated with TSE and contained 0.1% hydroxyquinoline. After two phenol extractions, the aqueous phase was treated with 2.5 % diethylpyrocarbonate (Bayer-Leverkusen, Germany) to inactivate ribonuclease and extracted twice with cold ether. The aqueous phase was then treated with >iO volume of 20% pot,assium acetate, pH 5.6, precipitated with 2.5 volumes of ethyl alcohol, and stored at -20” until use. For sedimentation in sucrose gradients the RNAs were analyzed directly, but prior to gel electrophoresis the HMW RNA was isolated by sedimentation in sucrose gradients (see below). The isolation of cellular ribosomal and transfer RNA will be described elsewhere (Obara et al., in preparation). Xeclimentation of RNA. Following alcohol precipitation the combined RNAs were taken up in 0.2 ml of TSE and layered on 5-20% linear sucrose gradients (4.8 ml) in TSE. Centrifugation was in an SW 50 rotor (Beckman Instruments Corp., Palo Alto, California) at 50,000 rpm for 55 min at 4”. The tubes were then punctured at the bottom and fractions were collected and assayed for radioactivity in a liquid scintillation spectrometer (Packard Instrument Corp., La Grange, Illinois). HMW RNA for gel electrophoresis was isolated from the gradients and reprecipitated in alcohol. Electrophoresis of RNA. XIixed agaroseacrylamide gels were prepared according t.o the procedure described by Peacock and Dingman (1968). Gels of 2.1% or 1.7 % acrylamide with 0.5 % agarose were polymer-
‘216
BOLOGNESI
ized at room temperature after mixing at 40”. The electrophoresis buffers used for the respective gels were also employed in the formation of the gels (Loening, 1969). Before application of the HMW RNA, a preliminary passage of current (60 min, 5 mA/gel) was employed to remove interfering ions. The RNA in 50-100 ,ul volumes of the respective buffers containing 7 % glycerol was then applied to the gels and electrophoresed at room temperature utilizing 7.5 V per centimeter of gel and 5 mA per gel. Following electrophoresis the gels were recovered and stained with methylene blue (Peacock and Dingman, 1968) or fractionated in l-mm slices, dissolved in 0.25 M piperidine by heating at 100” for 10 min, and subsequently assayed for radioactivity in 10 ml of Bray’s scintillation fluid. RESULTS
Sedimentation Properties of High Molecular Weight RNAs from Various Avian Tumor Virus Strains In order to obtain an optimal separation of the HMW RNAs it was necessary to sediment the molecules extensively and to fractionate the gradients into small samples. Two conditions were established to identify a significant difference in sedimentation: (1) the positions of the peak fractions had to be distinctly separated; and (2) the entire curves had to be displaced one from the other approximately the same number of fractions as were the positions of the peaks. To eliminate the possibility that the differences resulted from variations in handling of the RNAs, the respective viruses were mixed before extraction and the RNAs were sedimented in the same gradient. That labeling conditions were not the cause for the observed differences was ruled out by interchanging the isotopes. Using these criteria we set out to characterize the HMW RNAs of several strains of avian tumor viruses. The sedimentation properties of three HMW RNAs, a large (SRV-l), a medium (MAV-B), and a smalI (SRV-H) are shown relative to one another in Fig. 1. Although each pair of RNAs was cosedimented, the trials were performed in different experiments and the fractionations
AND
GRAF
were not always identical. Therefore, the number of fractions which separate the peaks do not reflect the absolute differences in sedimentation rates of the respective RNAs. For this, more precise estimates were performed and are discussed below. The RNTAs of the viruses studied seem to fall into three broad classes (Table 1) and the experiments described in Fig. 1 are representative examples. However, it is conceivable that the distribution of the HMW RNAs might be more complex. Of interest is that sarcoma virus RNAs are found which fall into each class and that one exists (SRV-H) which sediments slower than those of two leukosis strains (MAV-B and RAV50). From our studies it did not appear as though sedimentation properties of the RNAs could be directly correlated with serological properties of the viruses which determine their classification with regard to subgroup. Furthermore, it can be seen that viruses thought to be oncogenic in mammals, namely SRV-G and SRV-H, are present in the larger and smaller size classes. Whereas a variation in sedimentation behavior in most instances implies differences in molecular weight, it might also arise from conformational differences in the molecules, possibly resulting from the respective base compositions. It was thus of interest to employ other techniques to resolve t,his question. Analysis of the HMW RNAs by Gel Electrophoresis When the HMW RNAs of SRV-1 and SRV-H were examined on acrylamide gels, qualitatively similar differences to those obtained by sedimentation analysis were observed with respect to the relative rates of migration (Fig. 2). The electrophoresis was performed in mixed acrylamide-agarose gels which had been tested for their ability to separate RNAs of known molecular weight. In such gels a linear relationship was obtained with RNAs having a molecular weight below 2 X lo6 daltons (Fig. 3A). However, it was noticed that the position of the HMW RNA of AMV was far displaced from the line in that it migrated as a molecule having a lower molecular weight (Fig. 3A). This
AVIAN
TUMOR
VIRUS
RNAs
h \& I 10
1 20
I 0
I 10 Ffaabn
nwnlJ*r
I
1 20
10
20
FIG. 1. Sedimentation of viral HMW RNAs on sucrose gradients. RNAs from the respective virus strains labeled with uridine-3H or -32P were isolated with phenol and layered in pairs on 5-20’3, sucrose gradients in TSE. Centrifugation was for 55 min at 4’ in an SW 50 spinco rotor at 50,000 rpm. Fractions were taken and assayed for radioactivity. In each case, only that portion of the gradient representing the HMW RNA is indicated, and the volume of the fractions taken in A, B, and C was not the same. (A) SRV-lJH, O--O; SRV-H-SZP, .---0. (B) SRV-l-32P, O--C; MAV-B-3H, A---A. (C) A-A ; SRV-H-32P, 0-0. MAV-B-JH, TABLE DIFFERENCE
IN SEDIMENTATION
1
RATES OF HMW RNAs VIRUS STRAINS
FROM DIFFERENT
Size of RNAs according to sedimentation
AVIAN TUMOR
behavior
Type of virus Large Sarcoma Leukosis
SRV-1 SRV-G
Medium (A)a (D)
5 Capitals in parentheses indicate b See Materials and Methods.
MC-B MAV-B AMV RAV-50
the avian tumor
effect appears to be a peculiarity of the gels, but not of the HMW RNAs, as has also been observed by Peacock and Dingman (1968). The migration properties with respect to time of the HMW RNA of AR/IV in comparison to those of 27 S and 17 S
virus
Small (B) (B) (B)b (D)
SRV-H NC-SRV-H NC-SRV-1 RAV-7 RAV-1
(D) (D) (A) 0 (4
subgroup.
ribosomal RNA from chicken cells is shown in Fig. 3B. Whether or not gel electrophoresis may be used to determine molecular weight of RNA has not been strictly established. Recent studies with ribosomal RNAs on acrylamide
218
BOLOGNESI
Y,pratlon
Slice
AND
GRAF’
““WA.
FIG. 2. Electrophoresis
of RNAs on acrylamide gels. RNAs from SRV-1 labeled with uridineJH (O---C) and SRV-H labeled with 32P( O---e) were coelectrophoresed in E buffer on 2.1% acrylamide gels containing 0.57, agarose for 6 hr at room temperature employing 75 V and 5 mA per gel. The gels were then sliced and assayed for radioactivity.
gels have suggested that the technique may provide a reasonable estimate (Loening, 1969). However, it was pointed out that small overestimates are obtained with RNAs of very low G + C content due to more pronounced unfolding of such molecules in buffers normally used for electrophoresis. Such an effect could be eliminated when the electrophoresis was performed in buffers containing sufficient concentration of Mg ions which should induce the molecules to attain their most compact forms. We therefore performed the electrophoreses of two HRIW RNAs having different sedimentation properties under conditions where the molecules might be unfolded to a great extent (low salt) ; when unfolding is minimal (high salt); and finally, using i\Ig2+ to reduce the molecules to their most compact stat.es. The results of these electrophoreses (Fig. 4) indicated that similar differences in migration to those observed under standard conditions (high salt) (Fig. 4A) mere also present in electrophoreses employing low salt (Fig. 4B) or Mg”+ (Fig. 4C). The presence of Mg2+ appeared to cause substantial aggregation of the RNA as is evidenced by the presence of radioactivity in the higher molecular weight region of the gel. It is difficult to decide whether the slightly larger differences in the
FIG. 3. Molecular weight estimation by gel electrophoresis and migration properties of the HMW RNA. (A) Cellular ribosomal and transfer RNA together with 62 S AMV-RNA were coelectrophoresed on a mixed agarose-acrylamide gel (0.5$& and 2.17,, respectively) for 75 min with 75 V and 5 mA per gel. The gels were then stained with methylene blue, and the position of bands was measured. The molecular weight of the various RNAs were estimated by analytical ultracenkifugation (Obara et al., in preparation). (B) The respective RNAs were coelectrophoresed as in (A) on 1.7yo acrylamide gels containing 0.57n agarose for various time intervals and thereafter were stained with methylene blue.
migration of the respective HMW RNAs obtained by standard conditions or low salt in comparison to that obtained in the presence of il!Igz+ are of any significance. We conclude from t,hese results that the
AVIAN
TUMOR
VIRUS
RNAs
219
from the electropherograms because of the peculiarities of the gel system used, a reasonable estimate could be obtained from the sedimentation analyses. The sedimentation constant of the HMW RNA of AMV could be determined by analytical ultracentrifugation and was found to be 62 S (Obara et al., in preparation). Since the HMW RNA of MAV-B was indistinguishable from that of AMV in sediment’ation behavior it was possible to express the sedimentation difference between SRV-1 and MAV-B (about 3% under the conditions used) in terms of molecular weight (about 7 X lo5 daltons) utilizing Spirin’s formula (Spirin, 1963) : molecular weight = 1550. (sZO,~)~J. Similarly the difference between SRV-1 and SRV-H (about 6%) was estimated to be about 1.3 X lo6 daltons. It should be mentioned, however, that Spirin’s formula was derived using single-stranded ribosomal RNA and may not be applicable to the viral HMW RNA because of peculiarities of its struct’ure (Bader and Steck, 1969; Kakefuda and Bader, 1969). Analysis of the HMW RNAs Isolated from Sarcoma Viruses and Their Nonconverting Mutant Derivatives
FIG. 4. Electrophoresis of viral HMW RNAs under various ionic conditions. RNAs from 32Plabeled AMV (X--X) and uridine-3H labeled SRV-1 (O-0) were coelectrophoresed for 4.5 hr on 2.1ojo acrylamide gels containing 0.5% agarose at room temperature with 75 V and 5 mA per gel. (A) In low salt buffer. (B) In high salt (E buffer). (C) In low salt buffer containing 16 mM Mgz+.
differences in sedimentation and electrophoretic migration among the HMW avian tumor virus RNAs studied probably represent differences in molecular weight rather than conformational variations. Although it was not possible to estimate these differences
Treatment of SRV-1 with hydroxylamine resulted in the isolation of an infectious virus (NC-SRV-1) which had lost its ability to convert chicken cells but nevertheless demonstrated the capacity to interfere with the parent sarcoma strain. Rigid tests were performed to rule out the possibility that the nonconverting virus was originally present in the SRV-1 stock, and it was concluded that it arose through mutation (Graf et al., 1971). It was then of interest to determine whether the loss of converting ability was accompanied by a loss of genetic material. A comparative study of the HMW RNAs of NC-SRV-1 and SRV-1 revealed the patt’ern shown in Fig. 5. As one can see, a rather large difference in sedimentation behavior was observed (Fig. 5A), the HMW RNA of the mutant sedimenting slower. By comparison with SRV-H, it was determined that the RNA of NC-SRV-1 is the slowest sedimenting virus HMW RNA thus far tested. A difference in the rate of migration in polyacrylamide gel electrophoresis was also ob-
220
BOLOGNESI AND GRAF
5
10 Fmction
15 number Migration
c
Slice
Number
FIG. 5. Sedimentation and electrophoretic behavior of HMW RNA from a mutant nonconverting virus (NC-SRV-1) compared to that of the parent strain (SRV-I). (A) The RNAs of SRV-1 labeled with uridine-3H (O-O) and NC-SRV-1 labeled with azP(0-O) were sedimented as described in Fig. 1. (B) The RNAs were isolated after sucrose gradient centrifugation and coelectrophoresed on acrylamide gels as described in Fig. 2. (O---O), SRV-1; (a-e), NC-SRV-1.
tained (Fig. 5B). These results suggest a substantial reduction in the genome size (about 1.5 X lo6 daltons) as a result of hydroxylamine treatment. This was unexpected since this mutagen is thought mainly to induce point mutations in the RNA under the conditions used (Schuster, 1961), and just how the apparent deletion of the RNA oc: curred remains an open question. A similar nonconverting mutant was isolated from SRV-H, but in this case its RNA was indistinguishable from that of the parent virus both in electrophoretic migration and sedimentation behavior. In contrast to the NC-SRV-1 this agent (NC-SRV-H) might have resulted from a point mutation. DISCUSSION
The results of these studies suggest that there exist size differences in the high molecular weight RNAs from various avian tumor virus strains. Some evidence was obtained that conformational properties of the molecules were not responsible for the observed variations in rates of sedimentation or migration in electrophoresis. It was only possible to estimate the molecular weight differences between these RNAs utilizing the sed-
imentation analyses due to the apparent anomalies in the mixed agarose-acrylamide gels. It may be better to reexamine this question on pure acrylamide gels since the mobilities of the HMW virus RNAs in such systems appear to be lower relative to those of ribosomal RNA (unpublished observations). Nevertheless the 6 % difference in sedimentation in sucrose gradients between the RNAs of SRV-1 and SRV-H represents a rather large portion of nucleic acid. It would also be of interest to examine heat or DMSO-treated RNAs from these viruses to determine how the denatured RNAs behave in sedimentation and electrophoretic analyses. If the HMW RNA from these viruses is indeed an aggregate of three to four pieces of similar size (Duesberg, 1968) the differences in molecular weight between two HMW R.NAs could be either evenly distributed among the segments, or localized mainly in one or two subunits. Studies are in progress to distinguish between these alternatives. The differences in molecular weight between two virus HMW RNAs should also be reflected by the rates of inactivation of the viruses after treatment with mutagenic
AVIAN
TUMOR
agents. Preliminary experiment*s have revealed that the focus-forming ability of SRV-1 is inactivated more rapidly by hydroxylamine than that of SRV-H which is in accordance with the sedimentation and electrophoretic analyses of the respective HMW RNAs. Our studies revealed that the size differences observed among the HMW RNAs are not directly related to whether the agent is a leukosis or sarcoma virus, the virus subgroup, or whether or not the agent is capable of causing tumors in mammals. Leukosis virus RNAs were found which sedimented more rapidly than that of a sarcoma virus RNA (SRV-H). From our studies it also appeared that the chicken line or the phenotype of the host cell from which the virus is derived, C/O or C/A for MAV-B, for example, did not inAuence the size of the virus RNA. These observations are not inconsistent with the recent proposal by Toyoshima et al. (1970) that leukosis viruses might have originated from sarcoma viruses through loss of genetic material. Because of the wide variations in the sizes of the sarcoma virus RNAs, it is still conceivable that respective leukosis virus derivatives might have smaller RNAs. For at least one situation this appears to be the case. An infectious mutant of SRV-1 was isolated which had lost its converting ability and was found to have a H1\4W RNA smaller than that of the parent virus by about 1.5 X lo6 daltons. It is hopeful that the genetic material lost is indeed represented by functions necessary for cell conversion. When more is known about the replication of these R;IL’As, and if it becomes possible to isolate complementary nucleic acids, then cross-hybridization studies could be used to single out RNA sequences involved in the process of cell conversion. ACKNOWLEDGMENT The authors wish to thank Professor Dr. Werner Schgfer and Dr. Heinz Bauer for their support and interest in this work. Special thanks go to Miss G. Hiiper for invaluable technical assistance.
VIRUS
221
RNAs REFERENCES
J. P., and STECK, T. L. (1969). Analysis of the ribonucleic acid of murine leukemia virus. J. Viral. 4,454459. BAUER, H., and GRAF, T. (1969). Evidence for the possible existence of two envelope antigenic determinants and corresponding cell receptors for avian tumor viruses. liirology 37, 157-161. DUESBERG, P. H. (1968). Physical properties of Rous sarcoma virus RNA. Proc. Nat. Acad. Sci. U. S. 60, 1511-1518. DUFF, R. G., and VOGT, P. K. (1969). Characteristics of two new avian tumor virus subgroups. Virology 39, 1830. GOLD& A. (1970). Radio induced mutants of the Schmidt Ruppin strain of Rous sarcoma virus. Viral 40, 1022-1029. GRAF, T., BAUER, H., GELDERBLOM, H., and BOLOGNESI, D. P. (1971). Studies on the reproductive and cell converting abilities of avian sarcoma viruses. Virology, in press. KAKEFUD.4, T., and BADER, J. P. (1969). Electron microscopic observations on the ribonucleic acid of murine leukemia virus. J. Viral. 4, 460474. LANGLOIS, A. J., BONAR, R. A., RAO, P. R., D. P., BEARD, D., and BEARD, BOLOGNESI, J. W. (1966). BAI strain A avian (myeloblastosis) leukosis virus from myeloblast tissue culture. Proc. Sot. Exp. Biol. Med. 123, 286289. LOENING, U. E. (1969). The determination of molecular weight of ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 113, 131-138. MOSCOVICI, C., and VOGT, P. K. (1968). Effects of genetic cellular resistance on cell transformation and virus replication in chicken hematopoietic cell cultures infected with avian myeloblastosis virus (BAI-A) Virology 35, 487497. OBARA, T., BOLOGNESI, D. P., and BAUER, H. (1971). Ribosomal RNA in avian leukosis virus particles (in preparation). PEACOCK, A. C., and DINGMAN, W. C. (1968). Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agaroseacrylamide composite gels. Biochemistry 2,668674. ROBINSON, W. S., and BALUDS, M. A. (1965). The nucleic acid from avian myeloblastosis virus compared with the RNA from the Bryan strain of Rous sarcoma virus. Proc. Nat. Acad. Sci. U. S. 54, 1686-1692. SCHUSTER, H. (1961). The reaction of tobacco mosaic virus ribonucleic acid with hydroxylamine. J. Mol. Biol. 3, 447457. SPIRIN, A. S. (1963). Some problems concerning BADER,
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the macromolecular structure of ribonucleic acids. Progr. Nucl. Acid Res. Mol. Biol. 1, 301345. TOYOSHIMA, K., FRJIS, R. R., and VOGT, P. K. (1970). The reproductive and cell-transforming
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capacities of avian sarcoma virus B77: inactivation by UV-light. Virology 42, 163-170. VOGT, P. K., and ISHIZAKI, R. (1966). Patterns of viral interference in the avian leukosis and sarcoma complex. Virology 30, 368374.
43, 214-222 (1971)
Size Differences
among
the High Molecular
of Avian DAN1 Max-Plan&-Znstitut
fiir
RNA’S
Tumor Viruses
P. BOLOGNESI’ Virusforschung,
Weight
AND
THOMAS
Biologisch-medizinische
GRAF Abteilung,
Tiibingen,
Germany
Accepted October 1, 1970 Sedimentation analyses in sucrose gradients of high molecular weight RNAs from several strains of avian tumor viruses revealed considerable variations. Analysis in gel electrophoresis likewise revealed differences in the rate of migration and experiments with various ionic conditions suggested that the observed differences are probably not due to conformational effects in the molecules but reflect variations in molecular size. The maximum difference obtained corresponds to a molecular weight of about 1.3 X lo6 daltons. The observed differences were not correlated to (i) whether the agent was a sarcoma or leukosis virus or (ii) the virus subgroup, or (iii) the capacity of the virus to form tumors in mammals. It was shown, however, that mutagenization of a sarcoma virus to an infectious nonconverting virus was accompanied by a loss in molecular size of the RNA. The significance of these results is discussed. INTRODUCTION
In studies with the high molecular weight (HMW) RNAs from certain strains of avian tumor viruses, we noticed slight differences in the sedimentation behavior of the molecules. That such differences in sedimentation rates had not been previously observed may have been due to the virus strains studied. The sedimentation behavior of the HMW RNAs from a mixture of Rous sarcoma virus (RSV) and Rous-associated virus (RAV) in comparison to avian myeloblastosis virus (AMV), the latter also being a mixed strain (Moscovici and Vogt, 1968) revealed no differences (Robinson and Baluda, 1965). In the work reported here strains of known classification were employed, which in the case of sarcoma viruses were purified by single focus cloning. However, AMV, which in our laboratory consists mainly of subgroup B virus was also used for some analyses. 1 Recipient of Public Health Service Postdoctoral Fellowship No. l-F2-CA-38, 592-01 from the National Cancer Institute.
Avian RNA tumor viruses are classified according to some of their general properties. They may, for practical purposes, be subdivided into sarcoma and leukosis viruses depending on their ability to convert chicken fibroblasts. Viruses of both types can be further subdivided into four groups as determined by their envelope properties (Vogt and Ishizaki, 1966; Duff and Vogt, 1969). It became of interest to determine whether the sedimentation properties of the RNAs could be correlated with any of these functions. Since previous work (Gold& 1970; Toyoshima et al., 1970; Graf et al., 1971) suggested that nonconverting viruses might originate from sarcoma viruses by mutation, it was of particular interest to determine whether the genomes of sarcoma viruses were larger than those of leukosis viruses. Although the studies revealed that the latter is not a general case, it could be demonstrated that mutagenization of a sarcoma virus gave rise to an infectious agent which had lost its converting ability and whose RNA was considerably smaller than that of the parent sarcoma strain. 214
AVIAN MATERIALS
TUMOR
AND METHODS
Buffers TSE: 0.01 M Tris. HCl, 0.1 M NaCl, 0.001 M EDTA, pH 7.2. Electrophoresis Buffer (E buffer): 0.04 M TriseHCl, 0.02 M Naf acetate, 0.001 M EDTA, 0.1% SDS, pH 7.4. Low Salt Buffer: 0.03 M Tris.HCl, 0.016 M HCI, 0.0001 M EDTA, 0.1 % SDS, pH 7.8. Low-salt, Mgz+ buffer: 0.03 M Tris .HCl, 0.016 M HCl 0.0001 M EDTA, 0.02 M Al@+ acetate, 0.1% SDS, pH 7.8. Viruses ancl cells. The origins of the Schmidt Ruppin strain 1 of Rous sarcoma virus (SRV-l), of SRV-H, nonconverting SRV-1 (NC-SRV-l), NC-SRV-H, and RAV-1 were described in a separate communication (Graf et al., 1971). A Schmidt Ruppin virus obtained from Dr. Alice Gold6 (SRV-G) was also employed (Graf et al., 1971). Rous-associated virus 50 (RAV-50) and RAV-7 were kindly supplied by Dr. P. Ii. Vogt, Seattle, Washington. The MC-29 strain was obtained through the courtesy of Dr. A. J. Langlois, and the MC-B was derived by focus cloning and belongs to subgroup B (unpublished results). The BAI-A strain of AYIV was originally obtained from Dr. J. W. Beard, Durham, North Carolina. AMV was isolated from the plasma of leukemic chickens and in our laboratory consists mainly of virus from subgroup B. The isolation and propagation of MAV-B was reported earlier (Bauer and Graf, 1969). All viruses were grown on susceptible chicken embryo cells which were selected according to the classification of the virus used (Graf et al., 1971). Growth of radioactive virus. Labeling of virus grown in chicken embryo fibroblast cultures with uridine was performed in a modified Eagle medium in the presence of 6% heat inactivated calf serum. Incubation with 20 ~Ci/ml uridine-3H (20 mCi/mmole, Radiochemical Centre, Amersham, England) was for 18 hr at 37”. Before incorporation of 32P, the cells were washed 3 times with phosphate-free Eagle medium. They were then incubated at 37” for 18 hr with 100 &X/ml 32P-orthophosphate (carrier free, Radiochemical Centre,
VIRUS
RNAs
215
Amersham, England) in phosphate-free medium containing 6 % dialyzed calf serum. Myeloblast cultures (Langlois et al., 1966> to be labeled with 32Pwere similarly washed, but 20% dialyzed calf serum was employed during labeling. Isolation of RNA. For these experiments extensive purification of the viruses was not found to be necessary. Therefore two cycles of alternate high and low speed centrifugation were employed to recover the virus. The final pellets were taken up in TSE. buffer, combined in appropriate proportions according to radioactivity, and extracted with phenol and SDS at 4”. The phenol used was saturated with TSE and contained 0.1% hydroxyquinoline. After two phenol extractions, the aqueous phase was treated with 2.5 % diethylpyrocarbonate (Bayer-Leverkusen, Germany) to inactivate ribonuclease and extracted twice with cold ether. The aqueous phase was then treated with >iO volume of 20% pot,assium acetate, pH 5.6, precipitated with 2.5 volumes of ethyl alcohol, and stored at -20” until use. For sedimentation in sucrose gradients the RNAs were analyzed directly, but prior to gel electrophoresis the HMW RNA was isolated by sedimentation in sucrose gradients (see below). The isolation of cellular ribosomal and transfer RNA will be described elsewhere (Obara et al., in preparation). Xeclimentation of RNA. Following alcohol precipitation the combined RNAs were taken up in 0.2 ml of TSE and layered on 5-20% linear sucrose gradients (4.8 ml) in TSE. Centrifugation was in an SW 50 rotor (Beckman Instruments Corp., Palo Alto, California) at 50,000 rpm for 55 min at 4”. The tubes were then punctured at the bottom and fractions were collected and assayed for radioactivity in a liquid scintillation spectrometer (Packard Instrument Corp., La Grange, Illinois). HMW RNA for gel electrophoresis was isolated from the gradients and reprecipitated in alcohol. Electrophoresis of RNA. XIixed agaroseacrylamide gels were prepared according t.o the procedure described by Peacock and Dingman (1968). Gels of 2.1% or 1.7 % acrylamide with 0.5 % agarose were polymer-
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BOLOGNESI
ized at room temperature after mixing at 40”. The electrophoresis buffers used for the respective gels were also employed in the formation of the gels (Loening, 1969). Before application of the HMW RNA, a preliminary passage of current (60 min, 5 mA/gel) was employed to remove interfering ions. The RNA in 50-100 ,ul volumes of the respective buffers containing 7 % glycerol was then applied to the gels and electrophoresed at room temperature utilizing 7.5 V per centimeter of gel and 5 mA per gel. Following electrophoresis the gels were recovered and stained with methylene blue (Peacock and Dingman, 1968) or fractionated in l-mm slices, dissolved in 0.25 M piperidine by heating at 100” for 10 min, and subsequently assayed for radioactivity in 10 ml of Bray’s scintillation fluid. RESULTS
Sedimentation Properties of High Molecular Weight RNAs from Various Avian Tumor Virus Strains In order to obtain an optimal separation of the HMW RNAs it was necessary to sediment the molecules extensively and to fractionate the gradients into small samples. Two conditions were established to identify a significant difference in sedimentation: (1) the positions of the peak fractions had to be distinctly separated; and (2) the entire curves had to be displaced one from the other approximately the same number of fractions as were the positions of the peaks. To eliminate the possibility that the differences resulted from variations in handling of the RNAs, the respective viruses were mixed before extraction and the RNAs were sedimented in the same gradient. That labeling conditions were not the cause for the observed differences was ruled out by interchanging the isotopes. Using these criteria we set out to characterize the HMW RNAs of several strains of avian tumor viruses. The sedimentation properties of three HMW RNAs, a large (SRV-l), a medium (MAV-B), and a smalI (SRV-H) are shown relative to one another in Fig. 1. Although each pair of RNAs was cosedimented, the trials were performed in different experiments and the fractionations
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were not always identical. Therefore, the number of fractions which separate the peaks do not reflect the absolute differences in sedimentation rates of the respective RNAs. For this, more precise estimates were performed and are discussed below. The RNTAs of the viruses studied seem to fall into three broad classes (Table 1) and the experiments described in Fig. 1 are representative examples. However, it is conceivable that the distribution of the HMW RNAs might be more complex. Of interest is that sarcoma virus RNAs are found which fall into each class and that one exists (SRV-H) which sediments slower than those of two leukosis strains (MAV-B and RAV50). From our studies it did not appear as though sedimentation properties of the RNAs could be directly correlated with serological properties of the viruses which determine their classification with regard to subgroup. Furthermore, it can be seen that viruses thought to be oncogenic in mammals, namely SRV-G and SRV-H, are present in the larger and smaller size classes. Whereas a variation in sedimentation behavior in most instances implies differences in molecular weight, it might also arise from conformational differences in the molecules, possibly resulting from the respective base compositions. It was thus of interest to employ other techniques to resolve t,his question. Analysis of the HMW RNAs by Gel Electrophoresis When the HMW RNAs of SRV-1 and SRV-H were examined on acrylamide gels, qualitatively similar differences to those obtained by sedimentation analysis were observed with respect to the relative rates of migration (Fig. 2). The electrophoresis was performed in mixed acrylamide-agarose gels which had been tested for their ability to separate RNAs of known molecular weight. In such gels a linear relationship was obtained with RNAs having a molecular weight below 2 X lo6 daltons (Fig. 3A). However, it was noticed that the position of the HMW RNA of AMV was far displaced from the line in that it migrated as a molecule having a lower molecular weight (Fig. 3A). This
AVIAN
TUMOR
VIRUS
RNAs
h \& I 10
1 20
I 0
I 10 Ffaabn
nwnlJ*r
I
1 20
10
20
FIG. 1. Sedimentation of viral HMW RNAs on sucrose gradients. RNAs from the respective virus strains labeled with uridine-3H or -32P were isolated with phenol and layered in pairs on 5-20’3, sucrose gradients in TSE. Centrifugation was for 55 min at 4’ in an SW 50 spinco rotor at 50,000 rpm. Fractions were taken and assayed for radioactivity. In each case, only that portion of the gradient representing the HMW RNA is indicated, and the volume of the fractions taken in A, B, and C was not the same. (A) SRV-lJH, O--O; SRV-H-SZP, .---0. (B) SRV-l-32P, O--C; MAV-B-3H, A---A. (C) A-A ; SRV-H-32P, 0-0. MAV-B-JH, TABLE DIFFERENCE
IN SEDIMENTATION
1
RATES OF HMW RNAs VIRUS STRAINS
FROM DIFFERENT
Size of RNAs according to sedimentation
AVIAN TUMOR
behavior
Type of virus Large Sarcoma Leukosis
SRV-1 SRV-G
Medium (A)a (D)
5 Capitals in parentheses indicate b See Materials and Methods.
MC-B MAV-B AMV RAV-50
the avian tumor
effect appears to be a peculiarity of the gels, but not of the HMW RNAs, as has also been observed by Peacock and Dingman (1968). The migration properties with respect to time of the HMW RNA of AR/IV in comparison to those of 27 S and 17 S
virus
Small (B) (B) (B)b (D)
SRV-H NC-SRV-H NC-SRV-1 RAV-7 RAV-1
(D) (D) (A) 0 (4
subgroup.
ribosomal RNA from chicken cells is shown in Fig. 3B. Whether or not gel electrophoresis may be used to determine molecular weight of RNA has not been strictly established. Recent studies with ribosomal RNAs on acrylamide
218
BOLOGNESI
Y,pratlon
Slice
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““WA.
FIG. 2. Electrophoresis
of RNAs on acrylamide gels. RNAs from SRV-1 labeled with uridineJH (O---C) and SRV-H labeled with 32P( O---e) were coelectrophoresed in E buffer on 2.1% acrylamide gels containing 0.57, agarose for 6 hr at room temperature employing 75 V and 5 mA per gel. The gels were then sliced and assayed for radioactivity.
gels have suggested that the technique may provide a reasonable estimate (Loening, 1969). However, it was pointed out that small overestimates are obtained with RNAs of very low G + C content due to more pronounced unfolding of such molecules in buffers normally used for electrophoresis. Such an effect could be eliminated when the electrophoresis was performed in buffers containing sufficient concentration of Mg ions which should induce the molecules to attain their most compact forms. We therefore performed the electrophoreses of two HRIW RNAs having different sedimentation properties under conditions where the molecules might be unfolded to a great extent (low salt) ; when unfolding is minimal (high salt); and finally, using i\Ig2+ to reduce the molecules to their most compact stat.es. The results of these electrophoreses (Fig. 4) indicated that similar differences in migration to those observed under standard conditions (high salt) (Fig. 4A) mere also present in electrophoreses employing low salt (Fig. 4B) or Mg”+ (Fig. 4C). The presence of Mg2+ appeared to cause substantial aggregation of the RNA as is evidenced by the presence of radioactivity in the higher molecular weight region of the gel. It is difficult to decide whether the slightly larger differences in the
FIG. 3. Molecular weight estimation by gel electrophoresis and migration properties of the HMW RNA. (A) Cellular ribosomal and transfer RNA together with 62 S AMV-RNA were coelectrophoresed on a mixed agarose-acrylamide gel (0.5$& and 2.17,, respectively) for 75 min with 75 V and 5 mA per gel. The gels were then stained with methylene blue, and the position of bands was measured. The molecular weight of the various RNAs were estimated by analytical ultracenkifugation (Obara et al., in preparation). (B) The respective RNAs were coelectrophoresed as in (A) on 1.7yo acrylamide gels containing 0.57n agarose for various time intervals and thereafter were stained with methylene blue.
migration of the respective HMW RNAs obtained by standard conditions or low salt in comparison to that obtained in the presence of il!Igz+ are of any significance. We conclude from t,hese results that the
AVIAN
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VIRUS
RNAs
219
from the electropherograms because of the peculiarities of the gel system used, a reasonable estimate could be obtained from the sedimentation analyses. The sedimentation constant of the HMW RNA of AMV could be determined by analytical ultracentrifugation and was found to be 62 S (Obara et al., in preparation). Since the HMW RNA of MAV-B was indistinguishable from that of AMV in sediment’ation behavior it was possible to express the sedimentation difference between SRV-1 and MAV-B (about 3% under the conditions used) in terms of molecular weight (about 7 X lo5 daltons) utilizing Spirin’s formula (Spirin, 1963) : molecular weight = 1550. (sZO,~)~J. Similarly the difference between SRV-1 and SRV-H (about 6%) was estimated to be about 1.3 X lo6 daltons. It should be mentioned, however, that Spirin’s formula was derived using single-stranded ribosomal RNA and may not be applicable to the viral HMW RNA because of peculiarities of its struct’ure (Bader and Steck, 1969; Kakefuda and Bader, 1969). Analysis of the HMW RNAs Isolated from Sarcoma Viruses and Their Nonconverting Mutant Derivatives
FIG. 4. Electrophoresis of viral HMW RNAs under various ionic conditions. RNAs from 32Plabeled AMV (X--X) and uridine-3H labeled SRV-1 (O-0) were coelectrophoresed for 4.5 hr on 2.1ojo acrylamide gels containing 0.5% agarose at room temperature with 75 V and 5 mA per gel. (A) In low salt buffer. (B) In high salt (E buffer). (C) In low salt buffer containing 16 mM Mgz+.
differences in sedimentation and electrophoretic migration among the HMW avian tumor virus RNAs studied probably represent differences in molecular weight rather than conformational variations. Although it was not possible to estimate these differences
Treatment of SRV-1 with hydroxylamine resulted in the isolation of an infectious virus (NC-SRV-1) which had lost its ability to convert chicken cells but nevertheless demonstrated the capacity to interfere with the parent sarcoma strain. Rigid tests were performed to rule out the possibility that the nonconverting virus was originally present in the SRV-1 stock, and it was concluded that it arose through mutation (Graf et al., 1971). It was then of interest to determine whether the loss of converting ability was accompanied by a loss of genetic material. A comparative study of the HMW RNAs of NC-SRV-1 and SRV-1 revealed the patt’ern shown in Fig. 5. As one can see, a rather large difference in sedimentation behavior was observed (Fig. 5A), the HMW RNA of the mutant sedimenting slower. By comparison with SRV-H, it was determined that the RNA of NC-SRV-1 is the slowest sedimenting virus HMW RNA thus far tested. A difference in the rate of migration in polyacrylamide gel electrophoresis was also ob-
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BOLOGNESI AND GRAF
5
10 Fmction
15 number Migration
c
Slice
Number
FIG. 5. Sedimentation and electrophoretic behavior of HMW RNA from a mutant nonconverting virus (NC-SRV-1) compared to that of the parent strain (SRV-I). (A) The RNAs of SRV-1 labeled with uridine-3H (O-O) and NC-SRV-1 labeled with azP(0-O) were sedimented as described in Fig. 1. (B) The RNAs were isolated after sucrose gradient centrifugation and coelectrophoresed on acrylamide gels as described in Fig. 2. (O---O), SRV-1; (a-e), NC-SRV-1.
tained (Fig. 5B). These results suggest a substantial reduction in the genome size (about 1.5 X lo6 daltons) as a result of hydroxylamine treatment. This was unexpected since this mutagen is thought mainly to induce point mutations in the RNA under the conditions used (Schuster, 1961), and just how the apparent deletion of the RNA oc: curred remains an open question. A similar nonconverting mutant was isolated from SRV-H, but in this case its RNA was indistinguishable from that of the parent virus both in electrophoretic migration and sedimentation behavior. In contrast to the NC-SRV-1 this agent (NC-SRV-H) might have resulted from a point mutation. DISCUSSION
The results of these studies suggest that there exist size differences in the high molecular weight RNAs from various avian tumor virus strains. Some evidence was obtained that conformational properties of the molecules were not responsible for the observed variations in rates of sedimentation or migration in electrophoresis. It was only possible to estimate the molecular weight differences between these RNAs utilizing the sed-
imentation analyses due to the apparent anomalies in the mixed agarose-acrylamide gels. It may be better to reexamine this question on pure acrylamide gels since the mobilities of the HMW virus RNAs in such systems appear to be lower relative to those of ribosomal RNA (unpublished observations). Nevertheless the 6 % difference in sedimentation in sucrose gradients between the RNAs of SRV-1 and SRV-H represents a rather large portion of nucleic acid. It would also be of interest to examine heat or DMSO-treated RNAs from these viruses to determine how the denatured RNAs behave in sedimentation and electrophoretic analyses. If the HMW RNA from these viruses is indeed an aggregate of three to four pieces of similar size (Duesberg, 1968) the differences in molecular weight between two HMW R.NAs could be either evenly distributed among the segments, or localized mainly in one or two subunits. Studies are in progress to distinguish between these alternatives. The differences in molecular weight between two virus HMW RNAs should also be reflected by the rates of inactivation of the viruses after treatment with mutagenic
AVIAN
TUMOR
agents. Preliminary experiment*s have revealed that the focus-forming ability of SRV-1 is inactivated more rapidly by hydroxylamine than that of SRV-H which is in accordance with the sedimentation and electrophoretic analyses of the respective HMW RNAs. Our studies revealed that the size differences observed among the HMW RNAs are not directly related to whether the agent is a leukosis or sarcoma virus, the virus subgroup, or whether or not the agent is capable of causing tumors in mammals. Leukosis virus RNAs were found which sedimented more rapidly than that of a sarcoma virus RNA (SRV-H). From our studies it also appeared that the chicken line or the phenotype of the host cell from which the virus is derived, C/O or C/A for MAV-B, for example, did not inAuence the size of the virus RNA. These observations are not inconsistent with the recent proposal by Toyoshima et al. (1970) that leukosis viruses might have originated from sarcoma viruses through loss of genetic material. Because of the wide variations in the sizes of the sarcoma virus RNAs, it is still conceivable that respective leukosis virus derivatives might have smaller RNAs. For at least one situation this appears to be the case. An infectious mutant of SRV-1 was isolated which had lost its converting ability and was found to have a H1\4W RNA smaller than that of the parent virus by about 1.5 X lo6 daltons. It is hopeful that the genetic material lost is indeed represented by functions necessary for cell conversion. When more is known about the replication of these R;IL’As, and if it becomes possible to isolate complementary nucleic acids, then cross-hybridization studies could be used to single out RNA sequences involved in the process of cell conversion. ACKNOWLEDGMENT The authors wish to thank Professor Dr. Werner Schgfer and Dr. Heinz Bauer for their support and interest in this work. Special thanks go to Miss G. Hiiper for invaluable technical assistance.
VIRUS
221
RNAs REFERENCES
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the macromolecular structure of ribonucleic acids. Progr. Nucl. Acid Res. Mol. Biol. 1, 301345. TOYOSHIMA, K., FRJIS, R. R., and VOGT, P. K. (1970). The reproductive and cell-transforming
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capacities of avian sarcoma virus B77: inactivation by UV-light. Virology 42, 163-170. VOGT, P. K., and ISHIZAKI, R. (1966). Patterns of viral interference in the avian leukosis and sarcoma complex. Virology 30, 368374.