The low molecular weight RNAs of Rous sarcoma virus

42, 182-195 (1970)

VIROLOGY

The Low Molecular

Weight

RNAs

of Rous Sarcoma

Virus

I. The 4 S RNA J. MICHAEL DREW Department

BISHOP, WARREN E. LEVINSON, AND SULLIVAN, LOIS FANSHIER,

of Microbiology,

University of California

NANCY QUINTRELL, JEAN JACKSOllj

School of Meclicine,

San Francisco,

California

94123

Accepted May 27, 1970 Chromatography of Rous sarcoma virus RNA on methylated albumin-kieselguhr has allowed the identification of four classes of intravirion RNA: a substantial amount of 4 S RNA, a discrete 7 S component, small quantities of 18 and 28 S RNA, and the 70 S RNA of the viral genome. The 4 S RNA’s of the virus and its host cell have identical electrophoretic mobilities in 10% polyacrylamide gels and are methylated to the same extent. However, significant differences in nucleotide compositions are detectable. The virus apparently does not contain RNA corresponding to any of the other low molecular weight species of cellular RNA. The data are in accord with previous suggestions that RNA tumor viruses contain tRNA acquired from the host cell during assembly, but also indicate at least minor differences in composition between the cellular and viral 4 S RNA populations. The results of reconstruction experiments suggest that the viral 4 S RNA is not simply a contaminant derived from cellular debris. INTRODUCTION

Rous sarcoma virus and the related avian leukosis viruses cont’ain at least two forms of RNA (Robinson and Duesberg, 1968): (1) a high molecular weight, single-stranded RNA, which consists of several noncovalently joined subunits (Duesberg, 1968; Erikson, 1969; Montagnier et al., 1969) and is presumed to be the viral genome; and (2) a population of low molecular weight RNA, generally described as heterogeneous and assigned sedimentation coefficients ranging from 4 to 10 S. The latter material was originally considered to be composed primarily of fragments derived from the high molecular weight RNA by degradation. However, recent st’udies performed with avian myeloblastosis virus and mouse leukemia virus have shown that at least a portion of this low molecular weight RNA is methylated (Erikson, 1969), that it will hybridize to DNA of the host cell (Wollmann and Kirsten, 1968), and that it can be acylated with a variety of

amino acids (Bonar et al., 1967; Travnicek, 1969). These data have led to the generalalbeit still preliminary-conclusion that avian and murine RNA tumor viruses contain functional tRNAs, some or all of which are encoded in the host cell genome. To date, the low molecular weight RNA of RSVl has not been carefully studied. We have therefore undertaken to determine the composition and function of this RNA and, as a first step, have examined its electrophoretic mobility subsequent to any of several preparative fractionations. We conclude that preparations of purified RSV contain a homogeneous population of methylated 4 S RNA which is indistinguishable from host tRNA on the basis of chromatographic behavior and electrophoretic mobility, although differences in nucleotide com1 Abbreviations used: RSV, Rous sarcoma virus; AMV, avian myeloblastosis virus; MLV, mouse leukemia virus; SDS, sodium dodecyl sulfate; MAK, methyl-esterified albumin-kieselguhr. 182

ROUS

SARCOMA

VIRUS

4 S RNA

183

Schmidt-Ruppin st,rain from Dr. P. Vogt. position are detectable. The source and functional significance of this RNA are open to The latter strain was checked intermittently for contamination with leukosis virus by question, but the results of reconstruction experiments suggest that it is not simply a end-point dilution interference assays. Racontaminant derived from cellular debris. In dioisotopes were used at the following concentrations: uridine-3H, lo-20 pCi/ml; uriaddition, a minor RNA component, with dine-14C, 0.5 &i/ml; methylmethionine-3H, sedimentation velocity and electrophoretic 15 pCi/ml; and 3!P, 100 &X/ml. Infected mobility approximating that of a 7 S molecule, has been detected. Finally, several of cells, maintained in growth medium conthe principal low molecular weight RNA taining 2% calf serum, were exposed to the components of chick embryo cells have been appropriate isotopes for a single 12-hour analyzed. These are apparently identical to period unless ot#herwise indicated. The tissue those previously described in HeLa cells culture medium was t’hen harvested at 12(Darnell, 1968), and include the recently hour intervals over the course of the ensuing discovered 5.5 S molecule [originally denoted 3 days. Purification of virus. The procedure de7 S by Pene et al. (1968), but see Weinberg scribed by Duesberg et al. (Duesberg et al., and Penman (1968)] which is released from 1968) was used with minor modifications. 2s S ribosomal RNA by heat. All of the issues considered in this com- The virus contained in approximately 2 liters munication were examined with both the of tissue culture fluid was concentrated by precipitation with ammonium sulfate and Bryan st.rain of Rous sarcoma virus, which cont’ains an avian leukosis virus (Rousthen sedimented through 15 % (w/v) sucrose in STE to a cushion of 40 % (w/v) potassium associated virus 1)) and the Schmidt-Ruppin tartrat,e (1.305 g/ml) in 0.02 M TriseHCl, strain, which contains no leukosis virus. Where appropriat’e, the results of comparapH 7.4. The band of virus at t’he density tive analyses are given. In all other instances, interface was collected, diluted in STE, and the illustrated resu1t.s apply t,o both strains subjected to isopycnic centrifugation in preof virus. Thus, the conclusions reached are formed gradients of eit,her 25-65% sucrose pertinent to Rous sarcoma virus per se, and in STE, or 15-40% potassium tartrate in are not qualified by t,he possibility that some 0.02 M Tris.HCl, pH 7.4. In our hands, the of t)he findings are att)ributable solely to the tartrate gradients offer superior resolution, presence of Rous-associated virus. but’ the sucrose gradient#s are more convenient if the purified virus is to be extract,ed MATERIALS AND METHODS with phenol. Solutions and reagents. STE: 0.1 M NaClZmaZ centrifugation in sucrose density 1 mM EDTA-0.02 M Tris. HCI, pH 7.4. gradients. Centrifugation of RNA through Electrophoresis buffer: E buffer of Bishop gradients of 15-30% sucrose in STE was et al. (1967). Uridine-3H (20-30 Ci/mmole) ; performed in a Spinco model L-2 centrifuge, uridine-14C (50 mCi/mmole) and methylusing an SW 65 rotor. The conditions of methionine-3H (134 mCi/mmole) were all centrifugation are given with the individual purchased from Schwarz BioResearch. Or- figures. Collection of the gradients and prothophosphateJ2P, carrier free, was obtained cessing of the fractions for measurement of from New England Nuclear Corporation. radioactivity were carried out as previously SDS: sodium dodecyl sulfate; Matheson, described (Bishop and Koch, 1967). Coleman and Bell, Inc. Extraction of RNA. (a) From purified Propagation of cells and virus; labeling with virus. The final virus band was diluted in radioisotopes. The preparation of chick STE containing 1% 2-mercaptoethanol, and embryo fibroblast monolayer cultures, infecunlabeled cellular RNA (100 pg/ml) was tion of these with RSV, and bioassay of the added as carrier. After disruption of the virus with 1% SDS, three successive phenol virus were performed as previously described (Levinson, 1967). Bryan high titer strain of extractions were performed at, room temperaRSV was obtained from Dr. H. Rubin, the ture. RNA was precipitated by the addition

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of 2 volumes of absolute ethanol and storage at - 20” for 12 hours. The precipitate was then washed with a mixture of ethanol: STE (2: 1) at 4” and dissolved in STE. (b) From chick embryo fibroblasts. A solution of 1% SDS in 0.05 M sodium acetateeO.01 M EDTA, pH 5.0, was added to the cell monolayers in volumes sufficient to give cell concentrations of less than 1 X lo’/ ml. After 5 min of gentle rocking at room temperature, the viscous solution was removed from the cell culture dishes and subjected to three successive phenol extractions at 60”. RNA was then precipitated with ethanol as described above, Fractionation of RNA with 1 M NaCl. RNA was precipitated with 1 1M NaCl as previously described (Bishop and Koch, 1967). This procedure separates ribosomal and other high molecular weight RNAs from tRNA, but it is maximally effective only if performed at RNA concentrations of at least 1 mg/ml. All RNA preparations were therefore adjusted to this concentration, using unlabeled cellular RNA as carrier if necessary, prior to salt fractionation. Chromatography of RNA on MAK columns. Details of this procedure have been published (Bishop and Koch, 1967). The columns used in the present st’udy measured 0.9 X 15 cm, and contained 4 g of kieselguhr bed and 10 mg of methylated albumin. All solutions were buffered with 0.02 M Tris. HCl, pH 7.4. RNA was adsorbed to the columns in 0.4 M NaCl and eluted with linear gradients of 0.4 M to 1.6 M NaCl, formed with an ISCO Dialagrad (Instrumentation Scientific Company, Lincoln, Nebraska) and maintained at a flow rate of 20 ml/hr. Chromatography was performed at 35” to facilitate optimal recovery and resolution (Koch et al., 1969). Column eluates were monitored for absorbance at 260 nm with an ISCO UA-2 UV-recorder. Fractions of 2 ml were collected and examined for acid-precipitable radioactivity as previously described. Nucleotide compositions. Hydrolysis of “P-labeled RNA with 0.3 N KOH and electrophoretic separation of t’he resultant mononucleotides was performed according to Sebring and Salzman (1964). Preparation of low molecular weight cellular

ET AL.

RNAs. Several types of low molecular weight cellular RNA were used to standardize the electrophoretic analyses. These were prepared as follows. (a) 4 S (tRNA). Fractionation of steadystate labeled RNA with 1 M NaCl provides a convenient source of relatively pure 4 S RNA (see below, Fig. 4~). On some occasions, however, t,he RNA was further purified by chromatography on 1LIAK. (b) 5 S RNA. Unfractionated cellular RNA, labeled to steady state with uridine3H, was chromatographed on MAK in a manner similar to that illustrated in Fig. 2. These particular conditions are not optimal for the separation of 4 and 5 S RNA (the NaCl gradient is too steep). Consequently, a mixed population, somewhat enriched with the 5 S form, is obtained. Such material proved adequate for the present study. (c) 5.5 S RNA, also referred to as 7 S RNA (Pene et al., 1968) and 28 S-associated RNA (Weinberg and Penman, 1968), is normally hydrogen-bonded to the 28 S ribosomal RNA. It can be released by hot (SO’) phenol ext,raction, by heating purified RNA to 60-70” following extraction at lower temperatures, or by exposure of the RNA to other agents which can disrupt hydrogen bonds (Pene et al., 1968). We have used three separate procedures to prepare 5.5 S RNA for use as a marker in electrophoresis. In all instances, the initial extraction of RNA was performed at room temperature to prevent dissociation of the 5.5 S molecule from the 28 S RNA. (1) 28 S RNA was isolated by zonal centrifugation, heated to 65” for 5 min in E buffer, and used without further fractionation. (2) Alternatively, heated 28 S RNA was chromatographed on RIAK. The initial RNA to elute (at NaCl concentration of approximately 0.65 M) is a homogeneous population of 5.5 S RNA. This procedure is a convenient source of highly purified 5.5 S RNA (see below, Fig. 3c) and is applicable on a preparative scale. (3) High molecular weight RNA was prepared by precipitation with 1 M NaCl, then heated (65”, 5 min, 3 mM EDTA) and chromatographed on RIAK. The 5.5 S RNA obtained in this manner was moderately contaminated with 4 S and 5 S RNA (see below, Fig. 4b),

ROUS

SARCOMA

reflecting the incomplete removal of these forms by the 1 M NaCl fractionation. Puri$cation of R-17 bacteriophage. Crude lysates of E. coli K12 infected with R17 were kindly provided by Dr. H. Boyer. The phage was purfied according to Roblin (1968), except that RNase-treatment was omitted. Polyacrylamide gel electrophoresis. Electrophoresis of low molecular weight RNA in gels of polyacrylamide, cross-linked with ethylene diacrylate, was performed according to published methods (Loening, 1967; Bishop et al., 1967). The gels were polymerized in plexiglass tubing and used without a prerun. Eleetrophoresis was carried out at 5 mA/gel for 3.5 hours with 10 % gels, 4.75 hours with 2.25% gels. The gels were then frozen and cut into l-mm slices by stacked razor blades (Diversified Scientific Instruments, San Leandro, California). Slices were hydrolyzed in 1.Oml of concentrated ammonium hydroxide for 1 hour at room temperature, and counted in 10 ml of Bray’s solution prepared without methanol and ethylene glycol (Bray, 1960). RESULTS

Purification of RSV De--.-. I-- RNAs ~-J.- -L-. .- Analysis of RSV RNAs by zonal centrifugation is illustrated in Fig. la. In accord with previous reports (Robinson et al., 1965; Duesberg, 1968), t’wo major populations of RNA are apparent. The larger of these has a mean sedimentation coefficient of approximately 75-80 S2, as judged by its rate of sedimentation relative to that of R17 bacteriophage (Zinder, 1965). This RNA, with an estimated molecular weight of 1 X 107 (Robinson and Duesberg, 1968), is presumed to be the viral genome. Recently, it has been shown to consist of several noncovalently linked subunits, each with a molecular weight of approximately 3 X lo6 (Duesberg, * The high molecular weight RNA of RSV has been report,ed to have an SZC,,~ of 71 S, determined at 0.11 M NaCl in the analytical ultracentrifuge (Robinson and Duesberg, 1968). The present study was intended to give only relative sedimentation coefficients, and we attach no special significance to the higher value (75-80 S) obtained. For t,he sake of consistency, we shall subsequently refer to this RNA as 70 S.

VIRUS

185

4 S RNA

10

20 FRACTION

30

40

NUMBER

FIG. 1. Zonal centrifugation of RSV RNA. (a) Unfractionated RNA. RSV RNA, labeled with uridineJH (O---O), was analyzed in the presence of purified asp-labeled R-17 bacteriophage (0-O). Centrifugation: 50,069 rpm, 1% hours, 4”. (b) RNA fractionated with 1 M NaCl. Two preparations of RSV RNA, one labeled with uridine-3H the other with 32P, were subjected to 1 M NaCl fractionation. Portions of the precipitated 3H-RNA (.---0) and the soluble 32P-RNA (a- - -0) were mixed and analyzed. Centrifugation: 60,000 rpm, 1% hours, 4”.

1968; Montagnier et al., 1969). We have confirmed this observation (J. M. Bishop and D. Sullivan, unpublished observations), and will subsequently report on efforts to fractionate t.he subunit population. The 70 S RNA typically sediments in a relatively broad band (as in Fig. la), suggesting substantial heterogeneity of size. However, we consider this to be a reflection of aggregation, because the apparent heterogeneity can be largely eliminated by incubation at 37” for 30 min in the presence of 0.01 M EDTA (J. M. Bishop, unpublished observation). The second major component of RSV RNA is a low molecular weight population, the proportion of which (ca. 20%) is fairly constant in freshly prepared material. All

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BISHOP

RNA tumor viruses which have been examined to date contain such RNA, although the relative amounts reported have varied considerably, possibly as a function of the extent of degradation that may occur prior to or during extraction and/or storage. The high and low molecular weight RSV RNAs can be separated by NaCl fractionation, the 70 S RNA being insoluble, the lower molecular weight population, soluble (Fig. lb). The fractionation is virtually complete if the procedure is carried out under the conditions described above (Materials and Methods). MAK

Chromatography

Chromatography of RSV RNAs on MAK offers substantially better resolution than that obtained by zonal centrifugation (Fig. 2). At least four species of RNA are apparent. (1) A small amount of material elutes over the same range of NaCl concentration as do the ribosomal RNAs of the host cell. This conforms to previous reports that small amounts of host 18 S and 28 S ribosomal RNA are associated with the virions of RNA tumor viruses (Bonar et al., 1967; Robinson and Duesberg, 1968). The possible origin of these RNAs will be con-

ET AL.

sidered below. (2) A discrete population of RNA elutes at 0.75-0.78 M NaCl (RNA II in Fig. 2). To the best of our knowledge, this particular component of tumor virus RNA has not been detected previously. Preliminary analysis indicates that it is a singlestranded molecule with a sedimentation coefficient of 7 S, and that it is located within the virion. A complete report of these studies is in preparation. (3) The RNA which elutes subsequent to both ribosomal (18 and 28 S) and single-stranded poliovirus (35 S) RNAs is the 70 S RNA of RSV (RNA III in Fig. 2). Recovery of this RNA from the column varies considerably, generally ranging from 40 to 60%. This observation complies with the general experience that RNAs of high molecular weight can be difficult to recover from MAK (Koch et al., 1969). (4) Finally, a substantial amount of viral RNA (RNA I in Fig. 2) elutes at the same NaCl concentration as the 4 and 5 S RNAs of the host cell. This is the predominant low molecular weight constituent of RSV RNA, and is the object of the studies reported below. Recovery of such low molecular weight RNA from MAK is generally efficient and consist.ent, in contrast to that of high molecular weight RNAs. Consequently, the results of ?tIAK chromatography do not provide reliable est’imat,es of the relative amounts of RSV RNAs.

-15 i -10

-5

z 4 a. ::

-

10

20

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--7--m40

FRACTION

50

60

70

lion of the Gels 0 ;

80

NUMBER

FIG. 2. Chromatography of RSV RNA on MAK. 32P-labeled RSV RNA was ehromatographed in the presence of unlabeled cellular RNA (4~5 S, 18 S, 28 S) and 3H-labeled poliovirus RNA (35 S). The arrows denote the elution position of the cellular RNAs, identified by optical density, and of the 3H-labeled poliovirus RNA. Similar results were obtained with 3H-labeled RSV. l - - -0, 32P; X--X, NaCl gradient.

In an effort to define more precisely the low molecular weight constituents of RSV RNA, we turned to the use of electrophoresis in gels of 10% polyacrylamide. This procedure offers an except,ionally high degree of resolution and has been used for the separation of a variety of low molecular weight RNAs (Knight and Darnell, 1967; Nakamura et al., 1968; Weinberg and Penman, 1968). Migration of several standard types of cellular RNA through 10 % polyaerylamide gels is illustrat,ed in Fig. 3a. The following markers were employed: (1) 32P-4 S RNA which was obtained by 1 M NaCl fractionation of cellular RNA; and (2) The 5.5 S RNA released from 28 S ribosomal RNA by heating. An unident,ified species of RNA also

ROUS SARCOMA

lb

2b

3'0

40

50

60

70

187

VIRUS 4 S RNA

10

20

30

40

50

60

70

10 20 30 40 50 60 70 80

SUCE NUMBER 3. Electrophoresis of RSV low molecular weight RNA in 10% polyacrylamide gels. (a) Calibration of the gels. aHlabeled 28 S ribosomal RNA (a--@) in E buffer was heated at 65” for 5 min, cooled, and mixed with 32P-4S cellular RNA (O- - -0) obtained by 1 M NaCl fractionation. (b) Identification of 28 S-associated RNA. Aliquots of heated (60”, 5 min, E buffer) (+---a) and unheated (O-O) 3H-28 S RNA were electrophoresed separately, using a mixture of 3aP-labeled 4 and 5 S RNAs (@- - -0) as marker. The diagram is a composite of the data, superimposing the radioactivity profile of unheated 28 S RNA upon the results from the gel containing heated 28 ‘SRNA. (c) Electrophoresis of purified 5.5 S RNA. 3H-5.5 S RNA was purified by MAK chromatography of heated 28 S RNA (Materials and Methods, procedure number two) and electrophoresed with a pool of 32P-RNA containing predominantly 4 S RNA and a small amount of 5 S RNA. o-0, 3H; l - - -0, 32P. FIG.

appears after heating 28 S RNA. For the sake of convenience, we have designated this last RNA as “7 S.” It is an inconsistent finding to which we presently can attribute no significance. Two further electrophoretic analyses are also illustrated in order to validate our interpretation of the results presented in Fig. 3a. Electrophoresis of heated and unheated 3H28 S RNA, with 32P-labeled 4 and 5 S RNAs as markers, is shown in Fig. 3b. The low molecular weight RNA released from 28 S RNA by heat has an electrophoretic mobility dist.inct from that of 5 S RNA, and consistent

with its 5.5 S designation (Weinberg and Penman, 1968). The unhealed 28 S RNA sample contains no dissociated 5.5 S RNA, and no ot’her discrete population of low molecular weight RNA. Figure 3c illustrates the migration of purified 5.5 S RNA, prepared as described in Materials and Methods (procedure number 2). A pool of 4 and 5 S RNAs, eluted from MAK, was used as marker. The relative amount of 5 S RNA present in such preparations is small but unquestionably detectable (see inset). In summary, the foregoing data aecom-

188

BISHOP:ET

AL.

NaCl fractionation, and 5.5 S ribosomal RNA, partially purified by chromatography on MAK (Materials and Methods, procedure number 3). As noted before, the 5.5 S RNA prepared in this manner is moderately contaminated with 4 and 5 S RNAs. The results of these analyses indicate that RSV RNA I is composed solely of RNA with an electrophoretic mobility identical to that of cellular 4 S RNA. Electrophoresis of unfractimated RXV RNA. RSV RNA which had not been subjetted to any prior fractionation was electrophoresed in 10% polyacrylamide (Fig. 4~). The RNA which enters the gel is composed primarily of 4 S material. However, a small

plish two purposes. First, they illustrate the extent of resolution effected by the electrophoresis in 10% polyacrylamide gels. It seems reasonable to expect that RNA molecules with sedimentation coefficients as high as 8-10 S might enter these gels. Second, they confirm the previous reports concerning 5.5 S RNA (Pene et al., 1968; Weinberg and Penman, 1968), and demonstrate its existence in avian cells. Electrophoresis of RSV RNA I. The RSV RNA component which elutes first from MAN (RSV RNA I) was analyzed by electrophoresis (Figs. 4a and b). Two separate host cell RNAs were used as markers: 4 S tRNA, obtained from t,he supernatant of a r

b 5.5s

10

I 10

I 20

I 30

I 40

I 50

I 60

20 30

I 70

1

40

14s

50

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I 80

FIG. 4. Electrophoresis of RSV low molecular weight RNAs. (a) RSV RNA I with 4 S cellular RNA. were both isolated by MAK chromatog32P-RSV RNA I (@- - -0) and 3H-4 S cellular RNA (O---O) raphy. (b) RSV RNA I with 5.5 S cellular RNA. 32P-RSV RNA I was isolated by MAK chromatography. The 3H-5.5 S RNA was prepared according to Materials and Methods (procedure number three). Consequently, it is contaminated with both 4 and 5 S RNAs. a-0, 3H; a- - -0, 32P. (c) Unfractionated RSV RNA and 4 S cellular RNA. 3H-RSV RNA (a---a) was analyzed without any preliminary frac3zP-4 S RNA (a- - -0) was obtained from the supernatant of a 1 &f NaCl precipitation. tionation.

ROUS

SARCOMA

but measurable amount (note inset) of RNA migrates approximately in the position of the 7 S marker shown in Fig. 3a. This material corresponds to RSV RNA II (manuscript in preparation). Nucleotide Composition The preceding data indicate that the low molecular weight RNA of RSV consists primarily of 4 S RNA which is indistinguishable from cellular 4 S RNA on the basis

FRACTION

NUMBER

FIG. 5. Methylation of RSV RNA. Two preparations of unfractionated RSV RNA, one labeled with 3*P (@----a), the other with zH-labeled methylmethionine (O- - -a), were analyzed in the same sucrose gradient. Centrifugation was performed as in Fig. lb.

NUCLEOTIDE

20.4 19.6 24.8 p <

189

4 S RNA

of either chromatographic properties or electrophoretic mobility. This apparent identity was explored further by determining the nucleotide compositions of 4 S RNA from chick embryo cells, Bryan RSV, and SchmidtRuppin RSV (Table 1). The RNAs were prepared by chromatography on MAK, a procedure which yields highly purified viral 4 S RNA (Fig. 4a, b) and celiular 4 S Rh’A contaminated with barely detectable amounts of 5 S RNA (Fig. 5~). Despite an overall similarity in the results, the three populations of 4 S RNA exhibit statistically significant differences in their nucleotide compositions. The data for the 4 S RXA of chick cells and Bryan RSV are similar to previously published values, derived from RNA which had been isolated by zonal centrifugation (Robinson et al., 1965). To the best of our knowledge, the 4 S RNA of Schmidt-Ruppin virus has not previously been analyzed. Methylation of RXV RNA. The low molecular weight RNA of AMV can be substantially labeled by a donor of radioactive methyl groups (Erikson, 1969). We have made a similar observation in the case of RSV (Fig. 5). However, the results with AMV also suggested that the high molecular weight genome RNA may contain a small number of methyl groups. We have been unable to confirm this finding in t’he case of RSV (Fig. 5,) but the efficiency of methyl labeling in

TABLE 1 COMPOSITION OF CELLULAR AND RSV 4 S RNAsa U

Cellular Bryan Schmidt-Ruppin Bryan: cellular Schmidt-Ruppin: cellular Bryan:SchmidtRuppin

VIRUS

f 0.18 f 0.25 f 0.36 0.01

A

G 31.7 33.2 31.6 p <

f 0.33 ~JZ0.29 LIZ 0.29 0.001

p < 0.001

p > 0.8

18.0 19.5 18.3 p < p >

* 0.08 f 0.07 f 0.10 0.001 0.02

p < 0.001

p < 0.001

p < 0.001

C 29.9 27.7 25.7 p < p <

f 0.25 f 0.18 f 0.11 0.001 0.001

p < 0.001

a Cellular and RSV 4 S RNA’s were purified by MAK chromatography and analyzed for nucleotide composition. The upper portion of the table presents mean values (mole percentages of nucleotide) and standard errors, calculated from the results of twenty separate determinations, utilizing no less than three separate preparations of each form of RNA. For each nucleotide, the level of significance for the difference of the means has been determined by the t test and is expressed as the value of p (Bancroft, 19.57). These results are displayed in the lower portion of the table.

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BISHOP ET AL.

the RSV system is rather low. Consequently, we do not regard these results as conclusive. The nature of t’he methylated RNA and the extent of methylation were analyzed as follows. Infected cells were exposed simultaneously to methylmethionine-3H and uridine-14C. Released virus was periodically harvested over the course of 48 hours, then purified and its RNA extracted with phenol. At the conclusion of the harvesting, R1;A was also extracted from the cells. Viral RNA was fractionated by zonal centrifugation as in Fig. 5, and RNA isolated from the O-15 S region of the gradient was subjected to electrophoretic analysis (Fig. 6a). The migration of uridine-labeled RNA resembles t’hat of the viral RNA analyzed in the experiment of Fig. 4c. Virtually all t,he RNA is in a homogeneous 4 S populat,ion, but the more slowly migrating minor component (7 S) is again apparent. Labeled met,hyl groups are detectable only in the 4 S RNA, but the amount of minor component is so small that it precludes a definitive statement regarding the presence or absence of methylation. The cellular RNA was electrophoresed without prior fractionation (Fig. 6b). There is a substantial amount of uridineJ4C label in the 4 S RNA, and trace amounts are visible in the 5 S and 5.5 S regions of the gel (the RNA used in this experiment was phenol extracted at 60”, so no further treatment was necessary t,o release t,he 5.5 S species from the 28 S molecule). However, only the 4 S RNA contains detectable methyl label. This is not unexpected in view of the previous reports that, bot’h 5 and 5.5 S RNA contain few if any methyl groups (Forget and Weissman, 1968; Pene et al., 1968). The high molecular weight RNA, trapped at the origin of t,he gel, is abundantly methylat’ed. The ratio of methyl-3H label to uridine. 1% Iabe is 2.0 in t,he case of both viral and cellular 4 S RNA. A similar identity, involving different isotopes, has been reported by Erikson for AMV RNA (Erikson, 1969). The possible significance of these data will be discussed below.

I

I

10

20

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30

I

40

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,

I

60

70

I

80

SLICE NUMBER FIG. 6. Electrophoresis

of methyl-labeled RSV and cellular RNAs. (a) RSV RNA, labeled with uridine-1% (a- - -0) and 3H-labeled methylmethionine (@---a). Low molecular weight (O15 S) viral RNA was isolated by eonal centrifugation and then subjected to electrophoresis in 10% polyacrylamide gels. (b) Cellular RNA, labeled with uridine-1% (a- - -0) and 3H-labeled methylmethionine (@---a). RNA was extracted from the cells which had produced the virus used in (a), was analyzed by electrophoresis without any preliminary fractionation.

Contamination of Purifiecl RSV with Cellular RNA In an effort to explain the apparent association of ribosomal and 4 S RNAs with purified RSV, we have examined the possibility that these RNAs represent adventitious material derived from cellular debris. Uninfected cells were labeled with uridine-3H in a manner identical to that used for in.fected cells. The harvested tissue culture medium was then analyzed for material which, by virtue of its physicochemical properties, might persist as a contaminant

ROUS SARCOMA

VIRUS

4 S RNA

191

contained in the labeled virus purified from an equivalent volume of tissue culture medium (note the lo-fold difference in the two scales of Fig. 7). The nature of the RNAs in question was determined by electrophoretic analysis. Relatively long (200 mm) gels of 2.25% polyacrylamide were utilized in order to allow visualization of the full spectrum of viral RNAs in a single operation. Figure 8a illustrates the RNAs isolated from the preparation of labeled purified virus. All of the RNA forms previously identified by MAK chromatography (Fig. 2) are present, and no additional RNA species are apparent. The relative proportions of the various constituents of RSV RNA are given in Table 2 (column d). These data are more reliable than those obtained with MAK chromatography because of the erratic recovery of various RNA forms from MAK. Repeated electrophoretic analyses of this type have revealed only minor variations in the relative amount’s of the four RNA species found in preparations of RSV labeled to a steady state. Specifically, the values for the 4 S RNA have ranged from 20 to 25 %, those for 70 S genome RNA, from 65 to 70 %. FRACTION NUMBER The RNA contained in the cellular mateFIG. 7. Association of cellular RNA with RSV. rial which associates with unlabeled virus is Infected and uninfected cells were labeled for 12 hours with identical amounts of uridine-3H, and composed of the three major forms (28 S, 18 S, and 4 S) of cellular RNA (Fig. 8b) in the tissue culture medium then harvested at two approximately normal proportions (compare successive 4-hour intervals. In addition, medium was harvested from unlabeled, infected cells. columns a and b, Table 2). These proporEqual volumes of these were then processed tions contrast sharply with the relative through the standard purification procedure as amounts of t,he same species of RNA present follows: (a) Medium from labeled, uninfected cells in purified virus. In the latter instance, there (A-A); (b) a mixture of equal volumes of is a substantial excess of 4 S RNA over the medium from labeled, uninfected cells and from unlabeled, infected cells (a- - -0); and (c) ribosomal RNAs (column c, Table 2). On the medium from labeled, infected cells (O-O). basis of this disproportion, we conclude that The figure illustrates the final stage of purification, the presence of 4 S RNA in purified RSV viz., isopycnic centrifugation in performed gracannot be attributed simply to contaminadients of 25-65x sucrose (16 hours, 4’, 24,000 rpm, tion with cellular debris. In fact, the amount SW 25.3 rotor). Portions (50 ~1) of each 0.2 ml of radioactivity present in the adventitious fraction were taken for measurement of radioactivity, and the density of the indicated fractions 4 S RNA illustrated in Fig. 8b could not was determined by measuring the refractive index account for more than 10% of the total 4s The results of the three gradients are superimRNA found in virus isolated from an equiposed. The regions of each gradient which convalent volume of medium. Thus, 4 S RNA tained radioactivity were pooled and extracted with SDS-phenol as described in Materials and must be included selectively in preparat,ions Methods. of purified RSV.

throughout the standard virus purification. No such adventitious debris was observed when medium from normal cells was examined alone (Fig. 7), but labeled material derived from normal cells did associate with admixed unlabeled RSV and was not removed by the purification procedure (Fig. 7). This incidental label can account for approximately 10% of the radioactivity

192

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FIG. 8. Electrophoresis of RSV RNA and RNA from virus-associated cellular debris. The 3H-labeled RNA isolated from the gradients illustrated in Fig. 7 were electrophoresed in gels of 2.25% polyacrylamide (4% hours, 5 mA/gel, room temp.). Normal cellular RNA, labeled with 32P,was used in each case as an internal marker. (a) 3H-RNA extracted from the purified, labeled virus. (b) 3H-RNA extracted from the labeled cellular material which associates with unlabeled RSV. -, 3H cpm; -----, 3*P cpm. DISCUSSION

The RNAs of Rous Sarcoma V&us. We have utilized correlative analyses with MAE chromatography and polyacrylamide gel electrophoresis in an effort to delineate more clearly the individual RNA constituents of RSV. Four discrete classes of RNA are apparent (4 S, 7 S, ribosomal 18 S and 28 S, and 70 S), all of which survive RNase treatment of purified virus (J. M. Bishop and L. Fanshier, unpublished observation). In addition, the data specifically demonstrate several features of the low molecular weight RSV RNAs. First’, the overwhelming majority of this population is composed of homogeneous 4 S material. If, as previously suggested (Robinson et al., 1965; Bader and Steck, 1969), the RNA prepared from freshly purified virus includes degradation products, these are all larger than 10 S. Second, the 4 S RNA of RSV is methylated to approximately the same extent as that of the host

cell. The possible significance of this observation will be discussed below. Finally, there is a previously undetected minor RNA component with a sedimentation coefficient approximately 7 S. The nature of this RNA is presently unknown, but its sedimentation behavior, electrophoretic mobility, nucleotide composition, and capacity to hybridize with host. cell DNA will be reported in a subsequent publication. Methylation of RSV RNA. In general, the genomes of RNA viruses are not appreciably methylated (Erikson, 1969). Taking advantage of this fact, Erikson performed experiments in which he demonstrated that the low molecular weight RNA of AMV could be significantly labeled by a donor of radioactive methyl groups (Erikson, 1969). This result has two implications. First, it suggests that at least some of the low molecular weight viral RNA is not derived from degradation of the viral genome. Second, it

ROUS

SARCOMA

VIRUS

4 S RNA

193

methylated) became labeled under the conditions which we employed suggests that single carbon transfer did not introduce RNAa appreciable artifact into these experiments. Second, the ratios of 2H-methylmethionine RSV: All AdventiRSV: Cellular to uridine-14C label in various forms of RNA RNA Cellular tious Fraction RNA Species ConstitRNA cannot necessarily be regarded as reliable (4 uents (C) (4 (b) indicators of relative extents of methylation, because the intensity of labeling by uridine28 s 20% 6% 47% 48% 14Cwill depend upon the nucleotide composi18 S 4% 28% 15% 32% tion of the RNAs in question. However, the 4s 19% 25% 65% 20% similarity between the nucleotide composi7s 3% tion of the RSV RNA I and cellular 4 S 70 s 68% RNA (Table 1) probably obviates such a All computations are based on the data illuscriticism in the present instance. trated in Fig. 8. The radioactivity contained in The nature and signi$cance of RXV 4 S each form of RNA was totaled, and these sums RNA. Although the 4 S RNA of tumor were in turn used to calculate the relative proporviruses is now being extensively studied in a tions of various RNAs. Column a: Relative pronumber of laboratories, it has never been portions of the normal cellular RNA species (28 S, clearly established that this RNA is, in fact, 18 S, and 4 S) used as markers in the electroan integral virion component. Two alterphoreses shown in Fig. 8a and b. Column b: Relative proportions of the adventitious RNAs ananative explanations of its origin are generally lyzed in the electrophoresis illustrated by Fig. 8b. offered (Robinson et al., 1965; Bader and Column c: Relative proportions of apparent celluSteck, 1969). First, it could be derived from lar RNA species present in purified RSV RNA; the high molecular weight viral RNA by computations were performed with the data condegradation during purification and extractained in Fig. 8a, omitting the 70 S and 7 S forms. tion. Both the electrophoretic homogeneity Column d. Relative proportions of all RSV RNA of the 4 S RNA and the results of expericonstituents, as illustrated in Fig. 8a. All identiments with methyl labeling negate this fiable RNA species were included in the calculapossibility. Second, the 4 S RNA could tions. simply be a contaminant derived from cellular debris. We have performed reconraises the possibility that some or all of the struction experiments, the results of which low molecular weight RNA is not even cast considerable doubt upon this explanaencoded in the virus genome, but, rather, is tion (Figs. 7 and 8, and Table 2). However, composed of host RNA which has been in- it is clear that purified RSV, as prepared corporated into the virion during assembly. here, does contain detectable amounts of Further evidence in favor of this latter con- adventitious cellular RNA. The presence of clusion was obtained by examining t’he rela- small quantities of ribosomal RNA in preptive amounts of radioactivity incorporated arations of RNA tumor viruses has been into RNA from a 3H-methyl donor and from noted on a number of previous occasions orthophosphate-32P. The ratios of 3H:32P (Bonar et al., 1967; Robinson and Duesberg, were identical for the low molecular weight 1968). Our results provide a feasible explanaRNAs of AR/IV and its host cell. tion for these observations, although the We have performed similar experiments nature of the cellular debris which associates wit.11RSV and have obtained similar results. with RSV is presently indeterminate. PreHowever, our data are subject to two possumably, it is particulate material which sible criticisms. First, we did not take the either adsorbs to the surface of the virion or precautions necessary to suppress incorporabecomes trapped in viral aggregates. Treattion of radioactivity from methylmethionine ment of purified virus with RNase prior to into purines via single carbon transfer extraction of the RNA does not eliminate (Weinberg and Penman, 1968). The fact that (Bonar et al., 1967; neither 70 S viral RNA nor the 5 and 5.5 S these contaminants cellular RNAs (none of which are normally unpublished observations of the authors). TABLE

2

RELATIVEPROPORTIONSOFTHECONSTITUENTSOF RSV RNA AND ADVENTITIOUS CELLULAR

194

BISHOP

None of the available data provide any indication as to whether or not the 4 S RNA is actually a functionally significant virion constituent. In fact, there is no certain evidence that it is contained in biologically active virus particles, although we have been unable to demonstrate heterogeneity of the virion population by either zonal centrifugation or equilibrium centrifugation in density gradients of sucrose, potassium tartrate, and CsCl. There is also no indication as to whether or not the 4 S RNA of RSV represents functional tRNA as in the case of AMV (Bonar et al., 1967; Travnicek, 1969), nor is there definitive evidence regarding the possibility that some or all of it is encoded in the host cell DNA, as is apparently true for the 4 S RNA of MLV (Wollman and Kirsten, 1968). The latter issue is amenable to study with the usual hybridization techniques, but an examination for functional tRNA activity in RSV RNA may be logistically impractical for the present. The amount of RNA used in standard acylation assays (100 pg) (Taylor et al., 1968; Travnicek, 1969) is at least an order of magnitude greater than the quantities of RSV RNA I which can be practicably obtained with current preparative procedures. Whether or not there is functional tRNA in RSV, two further issues will require resolution: is the 4 S RNA contained in RSV acquired solely from the host cell, and, if so, is it simply a random sample or is it in some way unique? The data shown in Table 1 are of interest in this regard because they offer at least a crude indication that there are differences of composition among the cellular and viral 4 S RNA populations. It seems unlikely that the observed compositional differences are due to varying degrees of contamination with other species of RNA (or their degradation products). Such putative contaminants would have to possess both size and secondary structure identical to that of tRNA in order to coelute with it from MAK. In addition, the RNAs used in these analyses were composed of homogeneous populations of 4 S molecules, as determined by gel electrophoresis. In any event, this preliminary indication of differences between cellular and viral 4 S RNAs will

ET AL.

have to be confirmed by far more specific forms of analysis, such as those previously utilized to identify structural and/or conformational differences between normal cellular and virus-induced or virus-coded tRNAs (Kano-Sueoka and Sueoka, 1966; Subak-Sharpe et al., 1966). The preliminary results of such analyses performed on the lysyl-tRNA contained in AMY suggest that only one of the two detectable host species of this tRNA are incorporated into the virus (Travnicek and Riman, 1970). ACKNOWLEDGMENTS

We are indebted to Dr. Peter Duesberg for his frequent, generous advice, and to Dr. L. Levintow for his support, encouragement, and editorial

assistance. The work was supported by USPHS grants CA 10223 and AI 06862, USPHS training grant AIO0299, and grants from the Cancer Coordinating Committee of the University of California and the California Division of the American Cancer Society. REFERENCES BaDER, J. P., and STECK, T. L. (1969). Analysis of

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