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Electrophoretic analysis of the RNA of avian tumor viruses
VIROLOQY
60,
753-764 (1972)
Electrophoretic
Analysis
CHRISTINA Department
of Viral
of the RNA
M. SCHEELE
AND
of Avian
HIDESABURO
Tumor
Viruses
HANAFUSA
Oncology, The Public Health Research Institute of the City of New York, Inc., 455 First Avenue, New York, New York 10016 Accepted September 1, 1978
The heat-dissociated RNA of avian leukosis and sarcoma viruses of several subgroups was analyzed by polyacrylamide gel electrophoresis. Heterogeneous patterns obtained in early experiments appeared to be due primarily to RNA degradation during steps of RNA characterization by sucrose gradient centrifugation rather than procedures of virus purification, virus incubation at 37”, or RNA extraction. All the viruses studied contained a major RNA species with an estimated size between 2.5 and 2.8 X lo6 daltons and at least one minor RNA species of slight,ly smaller size. This suggests that if the genome of avian tumor viruses is indeed segmented, the subunits are of a similar size. The heat-dissociated RNA of RAV-2, RAV-60, R.AV-50, and Schmidt-Ruppin RSV (subgroup A) was generally less complex than that of SchmidtRuppin RSV (subgroup D). No significant difference was found in the RNA of RAV-2 grown in chicken cells that were positive or negative for the expression of latent viral genes (chf). However, in the absence of other known leukosis viruses, the pattern of heat-dissociated RNA of Bryan RSV grown in cells expressing chf functions was consistently broader than that of Bryan RSV grown in cells not expressing chf functions. INTRODUCTION
Chemical analysts show that avian leukosis and sarcoma viruses contain 1-2 % RNA by weight (Crawford and Crawford, 1961; Quigley et al., 1971). Robinson et al. (1965) first isolated and characterized the RNA of Rous sarcoma virus (RSV) and their studies revealed two single-stranded components : fast sedimenting, 60-70 S RNA and slow sedimenting 4-10 S RNA. The 60-70 S RNA had an estimated molecular weight near 1 X 10’ dalt.ons and regularly represented 70-50 % of the total RNA when virus was purified rapidly and its nucleic acid was isolated immediately by phenol extraction (Robinson et al., 1965; Robinson and Baluda, 1965). Duesberg (196Sb) further investigated the physical properties of RSV RNA by determining the effects of heat and dimethyl sulfoxide on the sedimentation coefficient and electrophoretic mobility of the fast sedimenting component. The hydrodynamic properties of the high molecular 753 Copyright All rights
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
weight RNA were changed by the treatments that dissociated hydrogen bonds. The major 36 S RNA species obtained by dissociation had an approximate molecular weight of 3.0 X lo6 daltons. This raised the possibility t’hat RSV RNA consisted of several RNA subunits rather than a single polynucleotide chain. Evidence in confirmation of this finding has been presented by a number of workers (Erikson, 1969; Monta,gnier et al., 1969; Bader and Steck, 1969). Genetic studies also support the view that, the genome of avian leukosis and sarcoma viruses is segmented. Duesberg and Vogt, (1970) and Martin and Duesberg (1972) found that nontransforming derivatives of a sarcoma virus lacked a particular RNA species, and they spcculatcd that the class a subunit contained the genctic information for ~11 transformation. Bolognclsi and Graf (1971) showed that mut,agenization of a sarcoma virus to an infectious nonconverting virus was accompanied by a loss in molecular
754
SCHEELE
AND HANAFUSA
size of the high molecular weight RNA. Recently, Vogt (1971b) and Kawai and Hanafusa (1972) have found high frequency recombination during mixed infection with avian tumor viruses. Influenza and reovirus which both also undergo rapid genetically stable reassortment of markers are known to contain segmented genomes (Hirst, 1962; Pons and Hirst, 1968; Duesberg, 1968a; Shatkin et al., 1968; Fields and Joklik, 1969). In a search for biochemical criteria to distinguish transforming from nontransforming viruses, we surveyed the heat-dissociated RNA of subgroup A, B, D, and E avian leukosis and sarcoma viruses to determine if characteristic properties relating to subgroup or transforming ability existed. We were particularly interested in studying RSV in the absence of other known leukosis viruses. Owing to its high resolving power, a polyacrylamide gel electrophoretic technique was used in this work. Since apparently normal chicken cells contain a genetic element termed chick cell-associated helper factor (chf) (H. Hanafusa et al., 1970), we also investigated the effect of chf on the pattern of heat-dissociated RNA of viruses grown in chf positive and negative chicken cells. MATERIALS
AND METHODS
Cell culture and viruses. The preparation of C/E (+), C/E (-) and quail tissue cultures has been described previously (Hanafusa and Hanafusa, 1968; Hanafusa, 1969). C/E(+) and C/E(-) chicken cells were formerly termed C/O(chf+) and C/O’(chf -), respectively (Scheele and Hanafusa, 1971). In this study the nontransforming viruses used were subgroup B RAV-2, subgroup D RAV-50, and subgroup E RAV-60; the transforming viruses used were SchmidtRuppin RSV of subgroups A and D (SRRSV A and SR-RSV D) and Bryan RSV (beta type). The growth and characteristics of SR-RSV have been reported (Hanafusa and Hanafusa, 1966; Kawai and Yamamoto, 1970). Bryan RSV (f) and RSV (-) (T. Hanafusa et al., 1972) were obtained from cultures of C/E(+) or C/E(-) cells following infection with Bryan RSV(-) in the presence of UV-Sendai virus. Transformed
C/E(+) cells produce RSV(f) with a titer generally of about 1 X 10’ FFU/ml (focusforming units) in quail cells. The amount of nontransforming helper virus (RAV-60) present in RSV(f) preparations was on the order of 1O-3 that of the transforming virus (H. Hanafusa et al., 1970). RSV(f) obtained as described above should be distinguished from RSV(R.AV-60) prepared by infection of transformed C/O cells with RAV-60 since the latter preparation contains RAV-60 in a quantity exceeding that of RSV(RAV-60). Transformed C/E( - ) cells produce RSV(-) which is noninfectious for all host cells tested thus far. However, the amount of physical RSV(-) particles released by transformed C/‘E( -) cells appears similar to that of RSV(f). Although noninfectious, the RSV( - ) particles do contain the entire RSV genome since they are capable of transforming cells once they are incorporated into cells by the aid of UV-Sendai virus (T. prepHanafusa et al., 1970a). The RSV(-) aration does not contain detectable amounts of RAV-60 (T. Hanafusa et al., 1972). Virus purification. Infected cells were exposed for a 16-hr interval to 3H-uridine (50 pCi/ml; 27 Ci/mmole) or i4C-uridine (8 &i/ml; 505 mCi/mmole) (Amersham Scarle, Illinois). The tissue culture fluid containing radioactive virus released from labeled cells was harvested at the intervals specified in the text and clarified by centrifugation at 8000 g for 10 min to remove cellular debris. Virus was purified from the supernatant fluid as described earlier (Scheele and Hanafusa, 1971) with the following modification: Before isopycnic banding, virus collected from the interface between the 20 and 60% sucrose solutions of the discontinuous gradient was diluted 3-fold with 0.1 X SSC (1 X SSC = 0.15 M NaCl and 0.015 1M sodium citrate) and treated for 30 min at 37” with 20 fig/ml pronase, selfdigested at 37” for 3 hr (Mizutani et al., 1970). Virus treated with pronase banded at the same density of 1.16 g/ml in the sucrose gradient as untreated virus. RNA extraction. RNA was routinely isolated from virus in the presence of yeast tRNA by addition of 1% sodium dodecyl sulfate (SDS), 0.05 % 2-mercaptoethanol
RNA
OF AVIAN
(J-ME), and an equal volume of phenol saturated with TEN buffer (0.01 M Tris, pH 7.4, 0.001 ill ethylenediaminetetraacetic acid, and 0.1 M NaCl) at room temperature. After two phenol extractions, RNA was precipitated at -20’ by the addition of two volumes of ethanol. Other RNA isolation procedures used in certain experiments were extraction with SDS in the presence of 0.5 % diethyl pyrocarbonate (DEPC) (Naftonc, New York) and extraction with phenol containing m-cresol and 8-hydroxyquinoline (Parish and Kirby, 1967). Polyacrylanaide gels. Polyacrylamide gels (2.2 %) cross-linked with ethylene diacrylatc w(‘rc prepared essentially by the method of Ducsberg (196Sa,b). The monomer solution cont,aining 30 g of deionized acrylamide (Duesberg and Rucckert, 1965) and 2.7 g of ethylene diacrylat,e in 100 ml of HZ0 was stored at -20” to prevent hydrolysis of the diacrylate (Duesberg and Vogt, 1970). To Lucite tubes (4.75 mm i.d., 14 cm long) closed at the bottom with parafilm and placed in a vertical position, agarose (0.08 %) was added to a height of 5 mm to prevent the gels from coming out of the tubes. Polymerization of 2.2 % monomer solution in E buffer (0.004 M Tris-acetate, pH 7.2; 0.002 M sodium acetat,e; 0.001 M cthylencdiaminetetraacetic acid) \vas catalyzed by final conccnt’rations of 0.08 % N , N , N’ , N’-tctramcthylcthylenediaminc and 0.04 %I freshly prepared ammonium pcrsulfatc. Columns of 75 mm were cast immediately and overlaid with water to form a flat surface and t’o prevent inhibition of the reaction by atmospheric oxygen. After it had stood for 1 hr at room temperature, the parafilm at the bottom of the tubes m-as replaced by Dacron mesh secured with a rubber band. The water was removed from the top, and the gels were prcclcctrophoresed at 50 V for 30 min in E buffer containing 0.2 % SDS. Heat-dissociation of virim RNA. A quantity containing no more than 200 pg of 4 S RNA carrier and at least 10,000 cpm of labclcd viral RNA was removed for ccntrifugation from the alcohol suspension stored at -20”. The amount containing t,he desired radioactivit,y was calculated from that prescnt in virus collected from the 15-50 %I linear
TTJMOR
VIRUSES
755
sucrose gradient before phenol-SDS extraction. After centrifugation and decantation, excess alcohol was removed by evacuation and the pellet was dissolved in 10 ~1 of E buffer containing 0.2% SDS and 10% glycerol. The virion RNA was heated at 70” for 3 min in a thin-walled l-ml conical tube (previously heated to inactivate any contaminating HNase) and immediately applied to preelectrophoresed gels from which overlying buffer had been removed. Electrophoresis. Samples of 10 or 20 ~1 were carefully overlaid with E buffer containing 0.2 % SDS and electrophorcsed at 2.5 mA/gel for 2 or 4 hr at room temperature. Three gels wcrc always run at the same time, and the gels were always used on the same day as they wclre prepared. During t’he 4 hr runs, a slight, expansion of about 10 mm was noticeable. Following electrophorcsis, gels were removed onto a sheet of parafilm by detaching the Dacron mesh and holding the tube verbically. The parafilm supporting the gel was positioned in a thin rectangular box so that the gel lay as freely as possible. After freezing at’ -70” for 15 min, the gel was placed with forceps on the cutting device of stacked razor blades. The l-mm slices obtained w(‘re removed from between the blades with forceps and placed in a scintillation vial cont,aining 0.5 ml of RNasc (10 ,.&ml). The vials were incubat’cd at, 37” overnight to rcl(~asc~isotope: from t,ho gcbls and 6 ml of Bray’s (1960) scintillation solution was added the next morning for drtermination of radioactivity. All values have been corrected for background and spillage of 3H or 14C. Preparation of cellular RNA markers. Primary mouse kidney cultures incubated at 37” for 3 days in minimal essential medium containing 10 % horse serum were labeled for 24 hr with 14C-uridinc (1 pCi/ml; 60 mCi/mmole). The cells were then further incubated for 5 hr in unlabeled medium to permit processing of ribosomal RNA prclcursors to 2X S and 18 S rRNA. After ivashing t,\vir(>, the monolaycrs \verct dissolvc>d in TEN buffrtr containing 1 % SDS and 0.05 % 2-JIE and extracted w&h phcnolSDS in the presence of 0.1%’ macaloid a.nd. xcast tRNA carriclr. From the alcohol preclplt~atc
756
SCHEELE
AND
HANAFUSA
stored at -2O”, quantities containing the desired amount of radioactivity were removed when required. Calculation. Since for a given gel composition there is a linear relationship between the electrophoretic mobility of a singlestranded RNA species and the logarithm of its molecular weight (Bishop et al., 1967; Peacock and Dingman, 1968; Gould et al., 1969), an average value for each fraction of a gel can be established by comparison with the marker peak of 28 S rRNA (1.65 X lo6 daltons) and of 18 S rRNA (0.65 X lo6 daltons) (Petermann and Pavlovec, 1966; McConkey and Hopkins, 1969). The major peak of heat-dissociated virion RNA is defined by the fraction containing the highest radioactivity. Once the value of heatdissociated RAV-2 RNA was determined, it was used for further approximations in conjunction with 2X S rRNA.
Parameters Vision
In early night was
scribed in Materials and Methods. The RNA extracted with phenol-SDS from virus banding at a density of 1.16 g/ml was then further characterized as 60-70 S material on a 5-20 % linear sucrose gradient. As seen in Fig. IA, the pattern of heat-dissociated RAV-2 RNA thus prepared was generally very broad and heterogeneous. The complexity of the electropherogram could be due to degradation of the viral genome during any of the above procedures. Thus, the effect of each process on the size of heat-dissociated RAV-2 RNA was examined in more detail. Virus and RNA purification. To test for RNA degradation during virus purification and RNA characterization, virus was sedimented onto the 60% sucrose cushion but not purified further. RNA was extracted directly from the material over the sucrose cushion and electrophoresed without separation of the 60-70 S fraction. It should be RESULTS noted here that electrophoretic analysis of native, unfractionated virion RNA revealed A$ecting th,e Size oj RAV-2 minor amounts of 4 S RNA and little, if any, RNA 28 S and 1s S RNA. Figure 1 illustrates the experiments, virus labeled over- differences in electropherograms of purified first extensively purified as de- RNA from maximally processed virus (A) B
400
200
FRACTION
NUMBER
FK. 1. Polyacrylamide gel electrophoresis of heat-dissociated RAV-2 RNA. (A) Purified R.NA from maximally processed virus labeled for 12 hr. (B) Unpurified RNA from minimally processed virus labeled for 17 hr. (C) Unpurified RNA from minimally processed virus collected hourly for 7 hr from cultures labeled for 17 hr. Samples of 10 ~1 were resolved by elect,rophoresis at 2.5 mA/gel for 2 hr at room temperature. The RNA migrated from left to right.
RNA
OF AVIAN
and unpurified RNA from minimally processed virus (B) upon heat-dissociation. The latter RNA appeared more homogeneous than the former. The reduction in the amount of material having a high electrophoretic mobility and the simplicity of the pattern obtained by heating the unpurified RKA of minimally processed virus support the view that RNA degradation can occur during virus purification, RNA characterization or both. Incubation of virus at 37”. Bader and Steck (1969) and Watson (1971) have already reported that prolonged incubation of murine leukemia virus at 37” can alter the pattern of RNA obtained by dissociation with heat, urea, formaldehyde, or dimethyl sulfoxide. Thus, virus isolated from culture fluids collected at 1 hr intervals could contain more native RNA than virus kept at 37” for a long period of time. To examine this possibility, RAV-2 infected cells were exposed continuously for 17 hr to 3H-uridine before the culture fluid was replaced with nonradioactive medium and then collcctcd at hourly intervals for 7 hr. Both preparations of RAV-2 were purified minimally and subjected to RKA extraction and elcctrophoretic analysis without separation of the 60-70 S component. The pattern of the unpurified RNA from minimally processed I-hr virus (Fig. 1C) was very similar to the pat’tern of the unpurified RNA from minimally processed virus lab&d overnight (Pig. 1B). This suggests that. incubation of virus at 37” may not have a significant affect on the pattern of heat-dissociated RNA, at least in the case of RAV-2. To fully clarify the situation, RNA ext,ractcd from maximally processed RAV-2 released at 37” within a short period of time was heat-dissociated and subjected to elcctrophoretic analysis without separation of the 60-70 S component by sucrose gradient centrifugation. The RAV-2 pattern prcRented in Fig. 2 is almost identical to that shown in Fig. 1C. Thus, virus purification appears to have lit#tlc, if any, effect on virion RNA. This implies that the main cause for t,he hetrrogencity of the hoat-dissociated RNA in Fig. IA IS RKA degradation during characterization of the viral RNA as 60-70
TUMOR
757
VIRUSES
RAV-2
‘I”
FRACTION
_ NUMBER
2. Polyacrylsmide gel electrophoresis of heat-dissociated RNA of nontransforming RAV-2 of subgroup B. The viral (0-a) and ribosomal (O-O) RNAs were prepared as described in Materials and Methods. The sample of 20 11 was coelectrophoresed at 2.5 mA/gel for 2 hr at room temperat,ure. The ribosomal RNA was included as a reference marker. FIG.
S. Increased handling of the viral RNA undoubtedly presents greater opportunities for RNA degradation by external RNases. RNA extraction procedures. The effect on virion RNA of extraction with SDS in the presence of the RNase inhibitor diethyl pyrocarbonatc (DEPC) (Naftone, New York) was examined. In addition, virus was treated or not treated with pronase to eliminate extraneously adsorbed cellular RNases before extraction with phenol-SDS or phenol containing m-cresol and 8-hydroxyquinoline (Parish and Kirby, 1967). The patterns of all of these heat-dissociated RNAs were similar and resembled those already presented in Fig. 1B. Neither pronase treatment of virus, the prcsencc of DEPC nor extraction with tho phenolic mixture had a noticeable effect on thr state of the RAV-2 heatdissociated RNA obtained.
75s
SCHEELE TABLE
1
ESTIMATED SIZS OF HEAT-DISSOCIATED RNA AVIAN LEUKOSIS AND SARCOMA VIRUSES Compared to rRNA’” Sub&mUP
A
B D E
Virus Number of experiments
SR-RSV A RAV-2 SR-RSV D RAV-50 B-RSV (f) RAV-60 B-RSV(-)c
ANi:, IiAN.4EUSA
5 33 1 2 12 2 8
OF
Compared F.A:qzb
Av;rge
Aveege
daltok)
daltons)
2.70 2.60 2.71 3.29 2.51 2.60 2.61
2.68 2.68 2.80 2.50 2.65 2.55
a The average size of the major peak of heatdissociated virion RNA was determined by comparison to cellular RNA marker as described in Materials and Methods. b The average size of the major peak of heatdissociated virion RNA was determined by comparison to heat-dissociated RAV-2 RNA in a separate experiment. c B-RSV(-) is distinguished from subgroup E virus on the basis of envelope properties.
The information yielded by these experiments permitted us to establish the following conditions as optimal for comparing t’he heat-dissociated RNA of avian leukosis and sarcoma viruses: (1) virus to be isolated from culture fluids of infected cells labeled COW tinuously for 16 hr bcforc collection twice at 4-hr intervals; (2) virus to be maximally purified by centrifugation on a discontinuous 20-60 % sucrose gradient, pronase treatment, and equilibrium sedimentation in 15-50 % sucrose; (3) virion RNA to be extracted with phenol-SDS and analyzed directly by polyacrylamide gel electrophoresis without fractionation by sucrose gradient centrifugation. Comparison of Nontransforming
Viruses
The patterns of heat-dissociated RNA presented in Fig. 1 indicate that RAV-2 contains a single major RNA peak and a minor component phoretic mobility
of slightly (fractions
higher
elect,ro-
13 and 19, respectively, in Fig. 1C). As seen in Fig. lB, the minor component (fraction 30) was sometimes
demonstrable
only
as a leading
shoulder of the major peak (fraction 25). When cellular ribosomal RNA was used as a reference standard, as in Fig. 2, the estimated size of the RNA in the major peak was 2.6 f 0.3 X lo6 daltons. This value was calculated from 24 of 33 experiments (Table l), extremely high and low determinations being omitted. It has not been established whether t’he major peak represents a normal distribution of a single RNA species or is composed of several RNA molecules of slightly different molecular weight. In either case, our findings suggest that if the genome of RAV-2 is truly segmentled, t,hc subunits appear to be of very similar size. To see whether the RAV-2 pattern was characteristic of nontransforming viruses, the heat-dissociated RNAs obt’ained from RAV-50 of subgroup D and RAV-60 of subgroup E wrrc compared directly bo that of RAV-2 by electrophoresis for a longer period of time. As seen in Fig. 3, the patterns are generally the same. Each virus poss~ssc~s a major RNA species and at least one minor RNA form of slightly lower molecular weight. The RNA of both the subgroup D and E virus was slightly larger than that of the subgroup B virus. These are typical patterns regularly observed and RAV-50 appears somewhat more complex than RAV60 and RAV-2. In addition, Fig. 3 demonstrates t>hat the expression of chf in C/E(+) chicken cells appears to have no effect on the number or distribution of the RNA sprcitxs obtained from RAV-2 upon heat, diwociation. Comparison forming
of Transforming Viruses
to Nontrans-
Duesberg and Vogt (1970) and Martin and Duesberg (1972) have already shown that transforming viruses a,ppear more complex than nontransforming viruses in that transforming viruses contain a component heavier (subunit a) than the major species found in nontransforming viruses (subunit b). Figure 4 demonstrates that the heat-dissociated RNA obtained from SRRSV of subgroup D appears more heterogeneous than either SR-RSV A, RAV-50, RAV-60, or RAV-2. Alt’hough the molecular weight of the RNA in the major SR-RSV D
RNA OF AVIAN
TUMOR
759
VIRUSES I
I
RAV-2
RAV-50
300 1
RAV-60
/
1/
40
40
40 FRACTION
NUMBER
FIG. 3. Polyacrylamide gel electrophoreais of heat.-dissociated RNA of nontransforming viruses of subgroups B, D, and E. The RNA samples of 20 11 were prepared as described in Materials and Methods and coelectrophoresed at 2.5 mA/gel for 4 hr at room temperat,ure. The bar indicates t,he position of 28 S rRNA
40
40 FRACTION
NUMBER
FIG. 4. Polyacrylamide gel electrophoresis of heat-dissociated RNA subgroups A and D. Procedkire ident.ical to Fig. 3. RAV-2 heat-dissociat,ed as a reference marker.
and SR-RSV A peaks was similar (Table l), the overall pattern of SR-RSV D was much broader than that of SR-RSV A. The major RNA species of nontransforming RAV-50 of subgroup D is also about the same size as the major RNA species of transforming SRRSV D, but the transforming virus repro-
of t,ransforming TtNA (O--O)
SR-RSV of was inclthded
ducibly contained more heterogeneous pieces of large molecular weight RNA. The SR-RSV A pattern resembles that of RAV-2, RAV-60, and RAV-50 more closely than that of SR-RSV D. The ratio of nontransforming to transforming virus in both SR-RSV populations was low: In the case
760
SCHEELE
AND
of SR-RSV A, interfering virus was not detectable by end-point dilution and the ratio of nontransforming to transforming virus was 1:2 as determined by testing cloned cells (Vogt, 1971a); in the case of SR-RSV D, the ratio of nontransforming to transforming virus was 1:3 by the newly developed plaque assay technique (Kawai and Hanafusa, 1972). Although the small amounts of nontransforming virus present in both viral preparations may contribute to the SR-RSV patterns, it seems unlikely that spontaneously appearing nontransforming virus can account for the differences between SR-RSV A and SR-RSV D. Thus it may be that SR-RSV D is inherently more heterogeneous for some other reason. For example, if viral RNA were transcribed from viral DNA integrated into host cell DNA, small segments of host cell RNA could remain covalently attached to viral RNA as a result of an error in posttranscriptional processing similar to that reported 1970; for SV40 (Lindberg and Darnell, Tonegawa et al., 1970). or C/E(-) Growth of B-RSV in C/E(+) cb.icken cells has definite biological consequences: When grown in the former, infectious RSV(f) is produced; when grown in the latter, noninfectious RSV( - ) is produced (Vogt, 1967; Hanafusa and Hanafusa, 1968; T. Hanafusa et al., 1970a). The two types of
FRACTION
FIG. 5. Polyacrylamide
HANAFUSA
particles differ chemically in that RSV(-) lacks a major glycoprotein present in RSV(f) (Scheele and Hanafusa, 1971). In the electrophoretic analysis of heat-dissociated BRSV RNA (Fig. 5), the major peak of RSV(f) appeared slightly broader and more heterogeneous than that of RSV(-) in many cases. The meaning of this finding cannot be explained immediately. The results shoNll in Fig. 5 demonstrate that the pattern of transforming Bryan RSV RNA and nontransforming RAV-2 RNA is essentially indistinguishable. The heat-dissociated RNA of B-RSV(f) appears to be slightly smaller than that of B-RSV(-) and both transforming viruses seem slightly smaller than nontransforming RAV-60 of subgroup E (Table 1). This finding was confirmed by a direct comparison of the heat-dissociated RNAs of B-RSV(f) and B-RSV(-) to RAV-60 and B-RSV(f) t.o B-RSV(-) (Fig. 6). The Bryan RSV( - ) preparations used in this study contained no detectable infectious avian leukosis viruses and B-RSV(f) contained only trace amounts of RAV-60. Nevertheless, we still considered the possibility that ‘(noninfectious,” nontransforming virus could contribute to the B-RSV patterns. Nontransforming virus spontaneously arising in B-RSV preparations, if any, would be presumably polymerasr positive even
NUMBER
gel electrophoresis of heat-dissociated RNA of transforming B-RSV grown in cells resistant to subgroup E virus: B-RSV(f) from C/E(+) cells; B-RSV(-) from C/E(-) cells. Procedure identical to Fig. 3. RAV-2 heat-dissociated RNA (O---O) was included as a reference marker.
RNA
OF AVIAN
TUMOR
FRACTION
6. Polyacrylamide cedure identical to that FIG.
gel elect,rophoresis of Fig. 3.
TABLE
Virus
POl~eraSe activitya (cpm/ml)
Infectivity” (FFU/ml)
OF
SpeCifiC activity %I
SR-RSV B-RSV(f)
A
8.5 X 104 8.1 x 104
6.75 X 106 1.55 x 106
NUMBER
of heat-dissociated
2
POLYMERASE ACTIVITY AND INFECTIVITY SR-RSV A AND B-RSV(f)
0.126 0.077
a Virus was concentrated into a pellet by centrifugation and assayed for polymerase activity in the presence of 5 fig of poly(rA): (dT)r2--ld (5O:l). The reaction mixture contained: O.lYe Nonidet -p40; 5 pmoles Tris.HCl, pH 8.3; 0.6 pmole magnesium acetate; 2 @moles dithiothreitol; 6 pmoles NaCl; 0.002 pmole dTTP, and 162 pmoles 3H-TTP. The mixture was incubated at 37” for 1 hr and then the acid-insoluble radioactivity was determined. The values have been adjusted to cpm incorporated per milliliter of original virus suspension. b The average of two determinations. Serial lo-fold dilutions of the original virus suspension were assayed on quail cells.
though “defective” in t(he same sense that B-RSV is helper-dependent. Since the ratio of nontransforming to transforming virus in SR-RSV A was low, a comparison of BRSV(f) and SR-RSV A with regard to polymerase activity and infectivity should indicate the presence or absence of large amounts of “noninfectious”, nontransform-
761
VIRUSES
RNA
of B-RSV
and RAV-60.
Pro-
ing virus in B-RSV(f). As seen in Table 2, the specific activity of the polymerase per transforming unit for B-RSV(f) was less t,han that of SR-RSV A. This suggests that that B-RSV(f) does not contain excessive amounts of nontransforming, “defective” virus detectable by this test. DISCUSSION
The heat-dissociated RNA of avian leukosis and sarcoma viruses was examined by polyacrylamide gel electrophoresis. In this study, an important factor influencing the integrity of the RNA was handling of the RNA: Conventional centrifugation in a sucrose gradient seemed to provide an opportunity for RNA degradation by externally contaminating RNases. As described by Bader and Steck (1969), a small number of nicks in the viral RNA may not affect the sedimentation coefficient of 60-70 S but upon heating, t’he damaged RNA could give heterogeneous electrophoretic patterns. On the other hand, virus purification had little, if any, effect on t,he size of heat-dissociated RAV-2 RNA. In contrast to murine leukemia virus (Bader and Steck, 1969; Watson, 1971), prolonged incubation of virus at 37” also did not significantly alter the size of heated RNA. These findings suggest that RNA degradation by virion-associated enzymes does not necessarily occur extensively with the avian tumor viruses studied here.
762
SCHEELE
AND
When procedures that minimized RNA degradation were used, all the viruses gave similar electropherograms consisting of a single major peak and at least one minor component. Table 1 summarizes our findings on the estimated size of the major heatdissociated RNA component of avian leukosis and sarcoma viruses. The presence of a single major peak suggests that if 60-70 S RNA is primarily composed of three to four smaller subunit,s, they are all very similar in size. Some of the figures show t,hat even with the cautions employed, somewhat heterogeneous patterns were sometimes obtained. This could be due to a slight degradation of the RNA before or during the process of electrophorcsis. The argument that the multiple peaks represent better resolution of intrinsic RNA component8s seems unsound because the het,erogeneous peaks appear randomly and unreproducibly and moreover, are always prominent in the lower molecular weight region. We consider t’he simple, sharp patterns more reflective of the true distribution of heat-dissociated virion RNA. Bolognesi and Graf (1971) have reported that variations in the size of high molecular weight RNAs of avian tumor viruses cannot be correlated with virus subgroup or whether the agent is a leukosis or sarcoma virus. Similarly, the avian tumor viruses used in this study could not be clearly distinguished on the basis of their heat-dissociated RNAs as all seemed to contain a single major RNA peak of approximately the same molecular weight. For subgroup E, the major RNA species obtained by heat-dissociation of transforming B-RSV was slightly smaller than that of nontransforming RAV-60. Although the major species of SR-RSV D was similar to that of nontransforming RAV-50, transforming virus of subgroup D consistently contained material larger than that of nontransforming virus. It should also be noted that the heat-dissociated RNA of subgroup D viruses appeared in general more heterogeneous than that of subgroup A, B, or E viruses. Although the contribution of spontaneously arising nontransforming virus to the patterns of transforming virus is difficult to assess, the fraction of nontrans-
HANAFUSA
forming virus in the SR-RSV preparations used in this study was 25 to 33 %. Therefore, the patterns presented do primarily reflect the heat-dissociated RNA of transforming virus rather than nontransforming virus. However, we were unable to distinguish class a and b subunits as clearly as Duesberg and Vogt (1970) and MarOin and Ducsbcrg (1972). With SR-RSV D, the RKA peak containing the highest radioactivit’y could conceivably be considered as subunit b, with the heavier material comprising subunit a. But wit8h SR-RSV A and B-RSV, we were unable to different’iate subunits a and b. Although l\,Iartin and Duesbrrg (1972) also failed to demonstrate subunit a in B-RSV, they attributed their failure to t,he presence of excessive leukosis virus associated wit’h their B-RSV. In t,his study, however, the B-RSV preparations contained no or very minute amounts (< 0.1%) of leukosis virus and the specific activity of the polymcrase per transforming unit for B-RSV(f) suggests that the B-RSV preparations do not appear to contain “defective,” nontransforming virus in excess of t’ransforming virus. The development of a more refined technique with higher resolving power will hopefully provide better characterization of heat-dissociated RNA components. The expression of chf in C/E(+) cells had no detectable effect on the pattern of RAV-2 heat-dissociated RNA. But t’he major RNA species of B-RSV(f) grown in C/E(+) cells was consistently broader and more complex than that of B-RSV(-) grown in C/E( -) cells. One possibility that could account for the slight difference observed would be that viral specific RNA present in normal C/E(+) cells may contribute to the B-RSV(f) pattern. Although RAV-60 itself is biologically detected only in small amounts (T. Hanafusa et al., 1970a), the RNA of chf, the precursor of RAV-60, could be present within C/E(+) cells. Thus the intracellular pool of viral RNA in C/E(+) cells may be comprised of both B-RSV and chf elements. ACKNOWLEDGMENT The authors wish to thank Dr. Teruko Hanafusa for her contribution to some experiments and
RNA
OF AVIAN
Susan McRimmon, Lucy IXMauro, and Roni L. Bisdorfer for excellent technical assistance. This work was supported by U. 8. Public Health Service Research Grants CA-08747 and CA-12177 from the National Cancer Institute. CMS was supported by Damon Runyon Cancer Research Fellowship DRF-575-AT. REFISRRNCXS B.~nmr, J. P. (1970). Synthesis of the l<.NA of RNA-containing tumor viruses. Virology 49, 494-504. B.rumt, J. I’., and STIXE~? T. L. (1969). Analysis of the ribonucleic acid of murine leukemia virus. .I. C’irol. 4, 454-459. UISHOP, l>.H.L., CL.~YUROOI~, J. R..,and SPIEGELM.~N, S. (1967). Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Uol. Biol. 26, 373-387. and GRAE‘, T. (1971). Size IhLOON~XI, n. I’., differences among the high molecular weight RNA’s of avian tumor viruses. Virology 43,214222. Be.\y, (:. A. (1960). A simple efficient liquid scintillat,or for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. I, 279-285. CHAWFORD, L. V., AND CRAWFORD, E. M. (1961). The properties of Rous sarcoma virus purified by density gradient centrifugation. Virology 23, 227-232. I)UI<;SBRRG, P. II. (1968a). The RNA’s of influenza virus. Proc. Xat. Acad. Sci. U.S. 59, 930-937. ~)~I~:SUEHG, P. H. (1968b). Physical properties of ltous sarcoma virus RNA. Proc. 1\Tat. Acad. Sci. rr.,!%!60, 1511-1518. I)UBSHIZRG, P. II., and RUECKERT, It. R. (19G5). Preparative zone electrophoresis of proteins on polyacrylamide gels in 8 IIf urea. Anal. Biochem. 11, 342381. DUESBERG, P. H., and VOGT, P. K. (1970). Differences between the ribonucleic acids of transforming and non-transforming avian tumor viruses. Proc. -Vat. Acad. Sci. U.S. Gi, 1673lG80. ERIKSON, II. L. (1969). Studies on the RNA from avian myeloblastosis virus. Virology 3i, 124-131. FII:LDS, 1%.N., and JOKLX, W. K. (1969). Isolat,ion and preliminary genetic and biochemical characterization of temperature-sensitive mutan& of reovirus. virology 37. 335342. GOULD, H. J., PINDER, J. C., MATTHE:\VS, H. It., and GORDON, A. H. (1969). Fractionation of low molecular weight fragments of ribosomal and viral RNA by polyacrylamide gel electrophoresis. Anal. Biochem. 29, 1-21. HANAFUSA, H. (1965). Analysis of the defective-
TUMOII
VIRUSI~S
763
ness of Rous sarcoma virus. III. Determining influence of a new helper virus on the host to interference of range and suscept,ibility RSV. Virology 25, 248-255. HANAFUSA, H. (1969). Rapid transformation of cells by Rous sarcoma virus. Proc. Nat. Acad. Sci. U.S. 63, 318-325. HANAFUSA, H., and HANAFUSA, T. (1966). Determining factor in the capacity of Rous sarcoma virus to induce tumors in mammals. Proc. Nat. Acad. Sci. U.S. 55, 532-538. HANAFUSA, H., and HANAFUSA, T. (1968). Furt,her studies on RSV production from transformed cells. Virology 34, 630-636. HAXAFUSA, H., MIYAMOTO, T., and HANAFuSA, T. (1970). A cell-associated factor essential for formation of an infectious form of Rous sarcoma virus. Proc. Sat. Acad. Sci. 7r.S. 66, 314321. Iihivh~us.4, T., MIYAMOTO, T., and HhNAFUS.I, H. (1970a). A type of chick embryo cell that fails to support formation of infectious HSV. Virology 40, 55-64. HIINAFUSA, T., H.4N.1~us.4, H., and MIYAMOTO, T. (1970b). Recovery of a new virus from apparently normal chick cells by infection with avian tumor viruses. Proc. Nat. Acad. Sci. U.S. 67, 1797-1803. HAXAFUSA, T., HANAFUSA, H., MIYAMOTO, T., and FLEISSNI~R, E. (1972). Existence and expression of tumor virus genes in chick embryo cells. Virology 47, 47548’L. HIRST, G. K. (1962). Genetic recombination with Newcastle disease virus, polioviruses and influenza. Cold Spring Harbor Synrp. &ant. Biol. 27, 303-309. KA~AI, S., and HANAFUSA, H. (1972). Genetic recombination with avian tumor virus T’irology 49, 37-44. K.iwar, S., and ~AMAMOTO, T. (1970). Isolation of different kinds of nonvirus producing cells transformed by Schmidt-Ruppin strain (subgroup A) of Rous sarcoma virus. Jap. J. Exp. Med. 40, 243-256. LINDBERG, V., and DARNELL, J. E. (1970). SV 40specific RNA in the nucleus and polyribosomes of transformed cells. Proc. Nat. Acad. Sci. U.S. 65, 1089-1096. MCCONIII~JY, 11:. H., and HOPKINS, J. W. (1969). Molecular weights of some HeLa ribosomal RNA’s, J. Afol. Biol. 39, 545-550. ~~~AIWIN, U. S., and DUESBERG, P. H. (1972). The a subunit in the RNA of transforming avian tumor viruses. I. Occurrence in different virus strains. II. Spontaneous loss resulting in nontransforming variants. Virology 47, 494497. MIZUTANI, S., BOICTTIGER, D., and TEMIN, H. M. (1970). A DNA-dependent DNA polymerase
764
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AND HANAFUSA
and a DNA endonuclease in virions of Rous sarcoma virus. Nature (London) 228, 424427. MONTAQNIER, L., GOLDI%, A., and VIGIER, P. (1969). A possible subunit structure of Rous sarcoma virus RNA. J. Gen. FiroZ. 4,449452. PARISH, J. H., and KIRBY, K. S. (1967). An extension of the naphthalene disulphonate method for mammalian nucleic acids. Biochim. Biophys. Acta 142, 273-275. PEACOCK, A. C., and DINGMAN,
C. W. (1968). Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agaroseacrylamide composite gels. Biochemislry 7, 66%
674. PETERMANN, M. L., and PAVLOVEC, A. (1966). The
subunits and structural ribonucleic Jensen sarcoma ribosomes. Biochim.
acids of
Biophys. Acta 114, 264-276. PONS, M. W., and HIRST, G. K. (1968). Polyacryl-
amide gel electrophoresis of influenza virus RNA. Virology 34, 385-388. QUIQLEY, J. P., RIFKIN, D. B., and REICH, E. (1971). Phospholipid composition of Rous sarcoma virus, host cell membranes and other enveloped RNA viruses. Virology 46, 106-116. ROBINSON, W. S., and BALUDA, 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.
ROBINSON, W. S., PITKANEN,
A., and RUBIN, H. (1965). The nucleic acid of the Bryan strain of Rous sarcoma virus: Purification of the virus and isolation of the nucleic acid. Proe. Nat. Acad. Sci. U.S. 54, 137-144. SCHEELE, C. M., and HANAFUSA, H. (1971). Proteins of helper-dependent RSV. Virology 45, 401410. SHATKIN, A. J., SIPE, J. D., and LOH, P. G. (1968). Separation of ten reovirus genomes segments by polyacrylamide gel electrophoresis. J. ViroE. 2, 986-998. TONEGAWA, S., WALTER, G., BERNARDINI, A., and DULBECCO, R. (1970). Transcription of the SV40
genome in transformed cells and during lytic infection. Cold Spring Harbor Synap. Quad. Biol. 35, 823-831. VOGT, P. K. (1967). A virus released by “non-producing” Rous sarcoma cells. Proc. Nat. Acad. sci. U.S. 58, 801-808. VOGT, P. K. (1971a). Spontaneous segregation of NT viruses from cloned sarcoma viruses. Virology 46, 939-946. VOGT, P. K. (1971b). Genetically stable reassort-
ment of markers during mixed infection with avian tumor viruses. Virology 46,947-952. WATSON, J. D. (1971). The structure and assembly of murine leukemia virus: Intracellular viral RNA. Virology 45, 586-597.
60,
753-764 (1972)
Electrophoretic
Analysis
CHRISTINA Department
of Viral
of the RNA
M. SCHEELE
AND
of Avian
HIDESABURO
Tumor
Viruses
HANAFUSA
Oncology, The Public Health Research Institute of the City of New York, Inc., 455 First Avenue, New York, New York 10016 Accepted September 1, 1978
The heat-dissociated RNA of avian leukosis and sarcoma viruses of several subgroups was analyzed by polyacrylamide gel electrophoresis. Heterogeneous patterns obtained in early experiments appeared to be due primarily to RNA degradation during steps of RNA characterization by sucrose gradient centrifugation rather than procedures of virus purification, virus incubation at 37”, or RNA extraction. All the viruses studied contained a major RNA species with an estimated size between 2.5 and 2.8 X lo6 daltons and at least one minor RNA species of slight,ly smaller size. This suggests that if the genome of avian tumor viruses is indeed segmented, the subunits are of a similar size. The heat-dissociated RNA of RAV-2, RAV-60, R.AV-50, and Schmidt-Ruppin RSV (subgroup A) was generally less complex than that of SchmidtRuppin RSV (subgroup D). No significant difference was found in the RNA of RAV-2 grown in chicken cells that were positive or negative for the expression of latent viral genes (chf). However, in the absence of other known leukosis viruses, the pattern of heat-dissociated RNA of Bryan RSV grown in cells expressing chf functions was consistently broader than that of Bryan RSV grown in cells not expressing chf functions. INTRODUCTION
Chemical analysts show that avian leukosis and sarcoma viruses contain 1-2 % RNA by weight (Crawford and Crawford, 1961; Quigley et al., 1971). Robinson et al. (1965) first isolated and characterized the RNA of Rous sarcoma virus (RSV) and their studies revealed two single-stranded components : fast sedimenting, 60-70 S RNA and slow sedimenting 4-10 S RNA. The 60-70 S RNA had an estimated molecular weight near 1 X 10’ dalt.ons and regularly represented 70-50 % of the total RNA when virus was purified rapidly and its nucleic acid was isolated immediately by phenol extraction (Robinson et al., 1965; Robinson and Baluda, 1965). Duesberg (196Sb) further investigated the physical properties of RSV RNA by determining the effects of heat and dimethyl sulfoxide on the sedimentation coefficient and electrophoretic mobility of the fast sedimenting component. The hydrodynamic properties of the high molecular 753 Copyright All rights
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
weight RNA were changed by the treatments that dissociated hydrogen bonds. The major 36 S RNA species obtained by dissociation had an approximate molecular weight of 3.0 X lo6 daltons. This raised the possibility t’hat RSV RNA consisted of several RNA subunits rather than a single polynucleotide chain. Evidence in confirmation of this finding has been presented by a number of workers (Erikson, 1969; Monta,gnier et al., 1969; Bader and Steck, 1969). Genetic studies also support the view that, the genome of avian leukosis and sarcoma viruses is segmented. Duesberg and Vogt, (1970) and Martin and Duesberg (1972) found that nontransforming derivatives of a sarcoma virus lacked a particular RNA species, and they spcculatcd that the class a subunit contained the genctic information for ~11 transformation. Bolognclsi and Graf (1971) showed that mut,agenization of a sarcoma virus to an infectious nonconverting virus was accompanied by a loss in molecular
754
SCHEELE
AND HANAFUSA
size of the high molecular weight RNA. Recently, Vogt (1971b) and Kawai and Hanafusa (1972) have found high frequency recombination during mixed infection with avian tumor viruses. Influenza and reovirus which both also undergo rapid genetically stable reassortment of markers are known to contain segmented genomes (Hirst, 1962; Pons and Hirst, 1968; Duesberg, 1968a; Shatkin et al., 1968; Fields and Joklik, 1969). In a search for biochemical criteria to distinguish transforming from nontransforming viruses, we surveyed the heat-dissociated RNA of subgroup A, B, D, and E avian leukosis and sarcoma viruses to determine if characteristic properties relating to subgroup or transforming ability existed. We were particularly interested in studying RSV in the absence of other known leukosis viruses. Owing to its high resolving power, a polyacrylamide gel electrophoretic technique was used in this work. Since apparently normal chicken cells contain a genetic element termed chick cell-associated helper factor (chf) (H. Hanafusa et al., 1970), we also investigated the effect of chf on the pattern of heat-dissociated RNA of viruses grown in chf positive and negative chicken cells. MATERIALS
AND METHODS
Cell culture and viruses. The preparation of C/E (+), C/E (-) and quail tissue cultures has been described previously (Hanafusa and Hanafusa, 1968; Hanafusa, 1969). C/E(+) and C/E(-) chicken cells were formerly termed C/O(chf+) and C/O’(chf -), respectively (Scheele and Hanafusa, 1971). In this study the nontransforming viruses used were subgroup B RAV-2, subgroup D RAV-50, and subgroup E RAV-60; the transforming viruses used were SchmidtRuppin RSV of subgroups A and D (SRRSV A and SR-RSV D) and Bryan RSV (beta type). The growth and characteristics of SR-RSV have been reported (Hanafusa and Hanafusa, 1966; Kawai and Yamamoto, 1970). Bryan RSV (f) and RSV (-) (T. Hanafusa et al., 1972) were obtained from cultures of C/E(+) or C/E(-) cells following infection with Bryan RSV(-) in the presence of UV-Sendai virus. Transformed
C/E(+) cells produce RSV(f) with a titer generally of about 1 X 10’ FFU/ml (focusforming units) in quail cells. The amount of nontransforming helper virus (RAV-60) present in RSV(f) preparations was on the order of 1O-3 that of the transforming virus (H. Hanafusa et al., 1970). RSV(f) obtained as described above should be distinguished from RSV(R.AV-60) prepared by infection of transformed C/O cells with RAV-60 since the latter preparation contains RAV-60 in a quantity exceeding that of RSV(RAV-60). Transformed C/E( - ) cells produce RSV(-) which is noninfectious for all host cells tested thus far. However, the amount of physical RSV(-) particles released by transformed C/‘E( -) cells appears similar to that of RSV(f). Although noninfectious, the RSV( - ) particles do contain the entire RSV genome since they are capable of transforming cells once they are incorporated into cells by the aid of UV-Sendai virus (T. prepHanafusa et al., 1970a). The RSV(-) aration does not contain detectable amounts of RAV-60 (T. Hanafusa et al., 1972). Virus purification. Infected cells were exposed for a 16-hr interval to 3H-uridine (50 pCi/ml; 27 Ci/mmole) or i4C-uridine (8 &i/ml; 505 mCi/mmole) (Amersham Scarle, Illinois). The tissue culture fluid containing radioactive virus released from labeled cells was harvested at the intervals specified in the text and clarified by centrifugation at 8000 g for 10 min to remove cellular debris. Virus was purified from the supernatant fluid as described earlier (Scheele and Hanafusa, 1971) with the following modification: Before isopycnic banding, virus collected from the interface between the 20 and 60% sucrose solutions of the discontinuous gradient was diluted 3-fold with 0.1 X SSC (1 X SSC = 0.15 M NaCl and 0.015 1M sodium citrate) and treated for 30 min at 37” with 20 fig/ml pronase, selfdigested at 37” for 3 hr (Mizutani et al., 1970). Virus treated with pronase banded at the same density of 1.16 g/ml in the sucrose gradient as untreated virus. RNA extraction. RNA was routinely isolated from virus in the presence of yeast tRNA by addition of 1% sodium dodecyl sulfate (SDS), 0.05 % 2-mercaptoethanol
RNA
OF AVIAN
(J-ME), and an equal volume of phenol saturated with TEN buffer (0.01 M Tris, pH 7.4, 0.001 ill ethylenediaminetetraacetic acid, and 0.1 M NaCl) at room temperature. After two phenol extractions, RNA was precipitated at -20’ by the addition of two volumes of ethanol. Other RNA isolation procedures used in certain experiments were extraction with SDS in the presence of 0.5 % diethyl pyrocarbonate (DEPC) (Naftonc, New York) and extraction with phenol containing m-cresol and 8-hydroxyquinoline (Parish and Kirby, 1967). Polyacrylanaide gels. Polyacrylamide gels (2.2 %) cross-linked with ethylene diacrylatc w(‘rc prepared essentially by the method of Ducsberg (196Sa,b). The monomer solution cont,aining 30 g of deionized acrylamide (Duesberg and Rucckert, 1965) and 2.7 g of ethylene diacrylat,e in 100 ml of HZ0 was stored at -20” to prevent hydrolysis of the diacrylate (Duesberg and Vogt, 1970). To Lucite tubes (4.75 mm i.d., 14 cm long) closed at the bottom with parafilm and placed in a vertical position, agarose (0.08 %) was added to a height of 5 mm to prevent the gels from coming out of the tubes. Polymerization of 2.2 % monomer solution in E buffer (0.004 M Tris-acetate, pH 7.2; 0.002 M sodium acetat,e; 0.001 M cthylencdiaminetetraacetic acid) \vas catalyzed by final conccnt’rations of 0.08 % N , N , N’ , N’-tctramcthylcthylenediaminc and 0.04 %I freshly prepared ammonium pcrsulfatc. Columns of 75 mm were cast immediately and overlaid with water to form a flat surface and t’o prevent inhibition of the reaction by atmospheric oxygen. After it had stood for 1 hr at room temperature, the parafilm at the bottom of the tubes m-as replaced by Dacron mesh secured with a rubber band. The water was removed from the top, and the gels were prcclcctrophoresed at 50 V for 30 min in E buffer containing 0.2 % SDS. Heat-dissociation of virim RNA. A quantity containing no more than 200 pg of 4 S RNA carrier and at least 10,000 cpm of labclcd viral RNA was removed for ccntrifugation from the alcohol suspension stored at -20”. The amount containing t,he desired radioactivit,y was calculated from that prescnt in virus collected from the 15-50 %I linear
TTJMOR
VIRUSES
755
sucrose gradient before phenol-SDS extraction. After centrifugation and decantation, excess alcohol was removed by evacuation and the pellet was dissolved in 10 ~1 of E buffer containing 0.2% SDS and 10% glycerol. The virion RNA was heated at 70” for 3 min in a thin-walled l-ml conical tube (previously heated to inactivate any contaminating HNase) and immediately applied to preelectrophoresed gels from which overlying buffer had been removed. Electrophoresis. Samples of 10 or 20 ~1 were carefully overlaid with E buffer containing 0.2 % SDS and electrophorcsed at 2.5 mA/gel for 2 or 4 hr at room temperature. Three gels wcrc always run at the same time, and the gels were always used on the same day as they wclre prepared. During t’he 4 hr runs, a slight, expansion of about 10 mm was noticeable. Following electrophorcsis, gels were removed onto a sheet of parafilm by detaching the Dacron mesh and holding the tube verbically. The parafilm supporting the gel was positioned in a thin rectangular box so that the gel lay as freely as possible. After freezing at’ -70” for 15 min, the gel was placed with forceps on the cutting device of stacked razor blades. The l-mm slices obtained w(‘re removed from between the blades with forceps and placed in a scintillation vial cont,aining 0.5 ml of RNasc (10 ,.&ml). The vials were incubat’cd at, 37” overnight to rcl(~asc~isotope: from t,ho gcbls and 6 ml of Bray’s (1960) scintillation solution was added the next morning for drtermination of radioactivity. All values have been corrected for background and spillage of 3H or 14C. Preparation of cellular RNA markers. Primary mouse kidney cultures incubated at 37” for 3 days in minimal essential medium containing 10 % horse serum were labeled for 24 hr with 14C-uridinc (1 pCi/ml; 60 mCi/mmole). The cells were then further incubated for 5 hr in unlabeled medium to permit processing of ribosomal RNA prclcursors to 2X S and 18 S rRNA. After ivashing t,\vir(>, the monolaycrs \verct dissolvc>d in TEN buffrtr containing 1 % SDS and 0.05 % 2-JIE and extracted w&h phcnolSDS in the presence of 0.1%’ macaloid a.nd. xcast tRNA carriclr. From the alcohol preclplt~atc
756
SCHEELE
AND
HANAFUSA
stored at -2O”, quantities containing the desired amount of radioactivity were removed when required. Calculation. Since for a given gel composition there is a linear relationship between the electrophoretic mobility of a singlestranded RNA species and the logarithm of its molecular weight (Bishop et al., 1967; Peacock and Dingman, 1968; Gould et al., 1969), an average value for each fraction of a gel can be established by comparison with the marker peak of 28 S rRNA (1.65 X lo6 daltons) and of 18 S rRNA (0.65 X lo6 daltons) (Petermann and Pavlovec, 1966; McConkey and Hopkins, 1969). The major peak of heat-dissociated virion RNA is defined by the fraction containing the highest radioactivity. Once the value of heatdissociated RAV-2 RNA was determined, it was used for further approximations in conjunction with 2X S rRNA.
Parameters Vision
In early night was
scribed in Materials and Methods. The RNA extracted with phenol-SDS from virus banding at a density of 1.16 g/ml was then further characterized as 60-70 S material on a 5-20 % linear sucrose gradient. As seen in Fig. IA, the pattern of heat-dissociated RAV-2 RNA thus prepared was generally very broad and heterogeneous. The complexity of the electropherogram could be due to degradation of the viral genome during any of the above procedures. Thus, the effect of each process on the size of heat-dissociated RAV-2 RNA was examined in more detail. Virus and RNA purification. To test for RNA degradation during virus purification and RNA characterization, virus was sedimented onto the 60% sucrose cushion but not purified further. RNA was extracted directly from the material over the sucrose cushion and electrophoresed without separation of the 60-70 S fraction. It should be RESULTS noted here that electrophoretic analysis of native, unfractionated virion RNA revealed A$ecting th,e Size oj RAV-2 minor amounts of 4 S RNA and little, if any, RNA 28 S and 1s S RNA. Figure 1 illustrates the experiments, virus labeled over- differences in electropherograms of purified first extensively purified as de- RNA from maximally processed virus (A) B
400
200
FRACTION
NUMBER
FK. 1. Polyacrylamide gel electrophoresis of heat-dissociated RAV-2 RNA. (A) Purified R.NA from maximally processed virus labeled for 12 hr. (B) Unpurified RNA from minimally processed virus labeled for 17 hr. (C) Unpurified RNA from minimally processed virus collected hourly for 7 hr from cultures labeled for 17 hr. Samples of 10 ~1 were resolved by elect,rophoresis at 2.5 mA/gel for 2 hr at room temperature. The RNA migrated from left to right.
RNA
OF AVIAN
and unpurified RNA from minimally processed virus (B) upon heat-dissociation. The latter RNA appeared more homogeneous than the former. The reduction in the amount of material having a high electrophoretic mobility and the simplicity of the pattern obtained by heating the unpurified RKA of minimally processed virus support the view that RNA degradation can occur during virus purification, RNA characterization or both. Incubation of virus at 37”. Bader and Steck (1969) and Watson (1971) have already reported that prolonged incubation of murine leukemia virus at 37” can alter the pattern of RNA obtained by dissociation with heat, urea, formaldehyde, or dimethyl sulfoxide. Thus, virus isolated from culture fluids collected at 1 hr intervals could contain more native RNA than virus kept at 37” for a long period of time. To examine this possibility, RAV-2 infected cells were exposed continuously for 17 hr to 3H-uridine before the culture fluid was replaced with nonradioactive medium and then collcctcd at hourly intervals for 7 hr. Both preparations of RAV-2 were purified minimally and subjected to RKA extraction and elcctrophoretic analysis without separation of the 60-70 S component. The pattern of the unpurified RNA from minimally processed I-hr virus (Fig. 1C) was very similar to the pat’tern of the unpurified RNA from minimally processed virus lab&d overnight (Pig. 1B). This suggests that. incubation of virus at 37” may not have a significant affect on the pattern of heat-dissociated RNA, at least in the case of RAV-2. To fully clarify the situation, RNA ext,ractcd from maximally processed RAV-2 released at 37” within a short period of time was heat-dissociated and subjected to elcctrophoretic analysis without separation of the 60-70 S component by sucrose gradient centrifugation. The RAV-2 pattern prcRented in Fig. 2 is almost identical to that shown in Fig. 1C. Thus, virus purification appears to have lit#tlc, if any, effect on virion RNA. This implies that the main cause for t,he hetrrogencity of the hoat-dissociated RNA in Fig. IA IS RKA degradation during characterization of the viral RNA as 60-70
TUMOR
757
VIRUSES
RAV-2
‘I”
FRACTION
_ NUMBER
2. Polyacrylsmide gel electrophoresis of heat-dissociated RNA of nontransforming RAV-2 of subgroup B. The viral (0-a) and ribosomal (O-O) RNAs were prepared as described in Materials and Methods. The sample of 20 11 was coelectrophoresed at 2.5 mA/gel for 2 hr at room temperat,ure. The ribosomal RNA was included as a reference marker. FIG.
S. Increased handling of the viral RNA undoubtedly presents greater opportunities for RNA degradation by external RNases. RNA extraction procedures. The effect on virion RNA of extraction with SDS in the presence of the RNase inhibitor diethyl pyrocarbonatc (DEPC) (Naftone, New York) was examined. In addition, virus was treated or not treated with pronase to eliminate extraneously adsorbed cellular RNases before extraction with phenol-SDS or phenol containing m-cresol and 8-hydroxyquinoline (Parish and Kirby, 1967). The patterns of all of these heat-dissociated RNAs were similar and resembled those already presented in Fig. 1B. Neither pronase treatment of virus, the prcsencc of DEPC nor extraction with tho phenolic mixture had a noticeable effect on thr state of the RAV-2 heatdissociated RNA obtained.
75s
SCHEELE TABLE
1
ESTIMATED SIZS OF HEAT-DISSOCIATED RNA AVIAN LEUKOSIS AND SARCOMA VIRUSES Compared to rRNA’” Sub&mUP
A
B D E
Virus Number of experiments
SR-RSV A RAV-2 SR-RSV D RAV-50 B-RSV (f) RAV-60 B-RSV(-)c
ANi:, IiAN.4EUSA
5 33 1 2 12 2 8
OF
Compared F.A:qzb
Av;rge
Aveege
daltok)
daltons)
2.70 2.60 2.71 3.29 2.51 2.60 2.61
2.68 2.68 2.80 2.50 2.65 2.55
a The average size of the major peak of heatdissociated virion RNA was determined by comparison to cellular RNA marker as described in Materials and Methods. b The average size of the major peak of heatdissociated virion RNA was determined by comparison to heat-dissociated RAV-2 RNA in a separate experiment. c B-RSV(-) is distinguished from subgroup E virus on the basis of envelope properties.
The information yielded by these experiments permitted us to establish the following conditions as optimal for comparing t’he heat-dissociated RNA of avian leukosis and sarcoma viruses: (1) virus to be isolated from culture fluids of infected cells labeled COW tinuously for 16 hr bcforc collection twice at 4-hr intervals; (2) virus to be maximally purified by centrifugation on a discontinuous 20-60 % sucrose gradient, pronase treatment, and equilibrium sedimentation in 15-50 % sucrose; (3) virion RNA to be extracted with phenol-SDS and analyzed directly by polyacrylamide gel electrophoresis without fractionation by sucrose gradient centrifugation. Comparison of Nontransforming
Viruses
The patterns of heat-dissociated RNA presented in Fig. 1 indicate that RAV-2 contains a single major RNA peak and a minor component phoretic mobility
of slightly (fractions
higher
elect,ro-
13 and 19, respectively, in Fig. 1C). As seen in Fig. lB, the minor component (fraction 30) was sometimes
demonstrable
only
as a leading
shoulder of the major peak (fraction 25). When cellular ribosomal RNA was used as a reference standard, as in Fig. 2, the estimated size of the RNA in the major peak was 2.6 f 0.3 X lo6 daltons. This value was calculated from 24 of 33 experiments (Table l), extremely high and low determinations being omitted. It has not been established whether t’he major peak represents a normal distribution of a single RNA species or is composed of several RNA molecules of slightly different molecular weight. In either case, our findings suggest that if the genome of RAV-2 is truly segmentled, t,hc subunits appear to be of very similar size. To see whether the RAV-2 pattern was characteristic of nontransforming viruses, the heat-dissociated RNAs obt’ained from RAV-50 of subgroup D and RAV-60 of subgroup E wrrc compared directly bo that of RAV-2 by electrophoresis for a longer period of time. As seen in Fig. 3, the patterns are generally the same. Each virus poss~ssc~s a major RNA species and at least one minor RNA form of slightly lower molecular weight. The RNA of both the subgroup D and E virus was slightly larger than that of the subgroup B virus. These are typical patterns regularly observed and RAV-50 appears somewhat more complex than RAV60 and RAV-2. In addition, Fig. 3 demonstrates t>hat the expression of chf in C/E(+) chicken cells appears to have no effect on the number or distribution of the RNA sprcitxs obtained from RAV-2 upon heat, diwociation. Comparison forming
of Transforming Viruses
to Nontrans-
Duesberg and Vogt (1970) and Martin and Duesberg (1972) have already shown that transforming viruses a,ppear more complex than nontransforming viruses in that transforming viruses contain a component heavier (subunit a) than the major species found in nontransforming viruses (subunit b). Figure 4 demonstrates that the heat-dissociated RNA obtained from SRRSV of subgroup D appears more heterogeneous than either SR-RSV A, RAV-50, RAV-60, or RAV-2. Alt’hough the molecular weight of the RNA in the major SR-RSV D
RNA OF AVIAN
TUMOR
759
VIRUSES I
I
RAV-2
RAV-50
300 1
RAV-60
/
1/
40
40
40 FRACTION
NUMBER
FIG. 3. Polyacrylamide gel electrophoreais of heat.-dissociated RNA of nontransforming viruses of subgroups B, D, and E. The RNA samples of 20 11 were prepared as described in Materials and Methods and coelectrophoresed at 2.5 mA/gel for 4 hr at room temperat,ure. The bar indicates t,he position of 28 S rRNA
40
40 FRACTION
NUMBER
FIG. 4. Polyacrylamide gel electrophoresis of heat-dissociated RNA subgroups A and D. Procedkire ident.ical to Fig. 3. RAV-2 heat-dissociat,ed as a reference marker.
and SR-RSV A peaks was similar (Table l), the overall pattern of SR-RSV D was much broader than that of SR-RSV A. The major RNA species of nontransforming RAV-50 of subgroup D is also about the same size as the major RNA species of transforming SRRSV D, but the transforming virus repro-
of t,ransforming TtNA (O--O)
SR-RSV of was inclthded
ducibly contained more heterogeneous pieces of large molecular weight RNA. The SR-RSV A pattern resembles that of RAV-2, RAV-60, and RAV-50 more closely than that of SR-RSV D. The ratio of nontransforming to transforming virus in both SR-RSV populations was low: In the case
760
SCHEELE
AND
of SR-RSV A, interfering virus was not detectable by end-point dilution and the ratio of nontransforming to transforming virus was 1:2 as determined by testing cloned cells (Vogt, 1971a); in the case of SR-RSV D, the ratio of nontransforming to transforming virus was 1:3 by the newly developed plaque assay technique (Kawai and Hanafusa, 1972). Although the small amounts of nontransforming virus present in both viral preparations may contribute to the SR-RSV patterns, it seems unlikely that spontaneously appearing nontransforming virus can account for the differences between SR-RSV A and SR-RSV D. Thus it may be that SR-RSV D is inherently more heterogeneous for some other reason. For example, if viral RNA were transcribed from viral DNA integrated into host cell DNA, small segments of host cell RNA could remain covalently attached to viral RNA as a result of an error in posttranscriptional processing similar to that reported 1970; for SV40 (Lindberg and Darnell, Tonegawa et al., 1970). or C/E(-) Growth of B-RSV in C/E(+) cb.icken cells has definite biological consequences: When grown in the former, infectious RSV(f) is produced; when grown in the latter, noninfectious RSV( - ) is produced (Vogt, 1967; Hanafusa and Hanafusa, 1968; T. Hanafusa et al., 1970a). The two types of
FRACTION
FIG. 5. Polyacrylamide
HANAFUSA
particles differ chemically in that RSV(-) lacks a major glycoprotein present in RSV(f) (Scheele and Hanafusa, 1971). In the electrophoretic analysis of heat-dissociated BRSV RNA (Fig. 5), the major peak of RSV(f) appeared slightly broader and more heterogeneous than that of RSV(-) in many cases. The meaning of this finding cannot be explained immediately. The results shoNll in Fig. 5 demonstrate that the pattern of transforming Bryan RSV RNA and nontransforming RAV-2 RNA is essentially indistinguishable. The heat-dissociated RNA of B-RSV(f) appears to be slightly smaller than that of B-RSV(-) and both transforming viruses seem slightly smaller than nontransforming RAV-60 of subgroup E (Table 1). This finding was confirmed by a direct comparison of the heat-dissociated RNAs of B-RSV(f) and B-RSV(-) to RAV-60 and B-RSV(f) t.o B-RSV(-) (Fig. 6). The Bryan RSV( - ) preparations used in this study contained no detectable infectious avian leukosis viruses and B-RSV(f) contained only trace amounts of RAV-60. Nevertheless, we still considered the possibility that ‘(noninfectious,” nontransforming virus could contribute to the B-RSV patterns. Nontransforming virus spontaneously arising in B-RSV preparations, if any, would be presumably polymerasr positive even
NUMBER
gel electrophoresis of heat-dissociated RNA of transforming B-RSV grown in cells resistant to subgroup E virus: B-RSV(f) from C/E(+) cells; B-RSV(-) from C/E(-) cells. Procedure identical to Fig. 3. RAV-2 heat-dissociated RNA (O---O) was included as a reference marker.
RNA
OF AVIAN
TUMOR
FRACTION
6. Polyacrylamide cedure identical to that FIG.
gel elect,rophoresis of Fig. 3.
TABLE
Virus
POl~eraSe activitya (cpm/ml)
Infectivity” (FFU/ml)
OF
SpeCifiC activity %I
SR-RSV B-RSV(f)
A
8.5 X 104 8.1 x 104
6.75 X 106 1.55 x 106
NUMBER
of heat-dissociated
2
POLYMERASE ACTIVITY AND INFECTIVITY SR-RSV A AND B-RSV(f)
0.126 0.077
a Virus was concentrated into a pellet by centrifugation and assayed for polymerase activity in the presence of 5 fig of poly(rA): (dT)r2--ld (5O:l). The reaction mixture contained: O.lYe Nonidet -p40; 5 pmoles Tris.HCl, pH 8.3; 0.6 pmole magnesium acetate; 2 @moles dithiothreitol; 6 pmoles NaCl; 0.002 pmole dTTP, and 162 pmoles 3H-TTP. The mixture was incubated at 37” for 1 hr and then the acid-insoluble radioactivity was determined. The values have been adjusted to cpm incorporated per milliliter of original virus suspension. b The average of two determinations. Serial lo-fold dilutions of the original virus suspension were assayed on quail cells.
though “defective” in t(he same sense that B-RSV is helper-dependent. Since the ratio of nontransforming to transforming virus in SR-RSV A was low, a comparison of BRSV(f) and SR-RSV A with regard to polymerase activity and infectivity should indicate the presence or absence of large amounts of “noninfectious”, nontransform-
761
VIRUSES
RNA
of B-RSV
and RAV-60.
Pro-
ing virus in B-RSV(f). As seen in Table 2, the specific activity of the polymerase per transforming unit for B-RSV(f) was less t,han that of SR-RSV A. This suggests that that B-RSV(f) does not contain excessive amounts of nontransforming, “defective” virus detectable by this test. DISCUSSION
The heat-dissociated RNA of avian leukosis and sarcoma viruses was examined by polyacrylamide gel electrophoresis. In this study, an important factor influencing the integrity of the RNA was handling of the RNA: Conventional centrifugation in a sucrose gradient seemed to provide an opportunity for RNA degradation by externally contaminating RNases. As described by Bader and Steck (1969), a small number of nicks in the viral RNA may not affect the sedimentation coefficient of 60-70 S but upon heating, t’he damaged RNA could give heterogeneous electrophoretic patterns. On the other hand, virus purification had little, if any, effect on t,he size of heat-dissociated RAV-2 RNA. In contrast to murine leukemia virus (Bader and Steck, 1969; Watson, 1971), prolonged incubation of virus at 37” also did not significantly alter the size of heated RNA. These findings suggest that RNA degradation by virion-associated enzymes does not necessarily occur extensively with the avian tumor viruses studied here.
762
SCHEELE
AND
When procedures that minimized RNA degradation were used, all the viruses gave similar electropherograms consisting of a single major peak and at least one minor component. Table 1 summarizes our findings on the estimated size of the major heatdissociated RNA component of avian leukosis and sarcoma viruses. The presence of a single major peak suggests that if 60-70 S RNA is primarily composed of three to four smaller subunit,s, they are all very similar in size. Some of the figures show t,hat even with the cautions employed, somewhat heterogeneous patterns were sometimes obtained. This could be due to a slight degradation of the RNA before or during the process of electrophorcsis. The argument that the multiple peaks represent better resolution of intrinsic RNA component8s seems unsound because the het,erogeneous peaks appear randomly and unreproducibly and moreover, are always prominent in the lower molecular weight region. We consider t’he simple, sharp patterns more reflective of the true distribution of heat-dissociated virion RNA. Bolognesi and Graf (1971) have reported that variations in the size of high molecular weight RNAs of avian tumor viruses cannot be correlated with virus subgroup or whether the agent is a leukosis or sarcoma virus. Similarly, the avian tumor viruses used in this study could not be clearly distinguished on the basis of their heat-dissociated RNAs as all seemed to contain a single major RNA peak of approximately the same molecular weight. For subgroup E, the major RNA species obtained by heat-dissociation of transforming B-RSV was slightly smaller than that of nontransforming RAV-60. Although the major species of SR-RSV D was similar to that of nontransforming RAV-50, transforming virus of subgroup D consistently contained material larger than that of nontransforming virus. It should also be noted that the heat-dissociated RNA of subgroup D viruses appeared in general more heterogeneous than that of subgroup A, B, or E viruses. Although the contribution of spontaneously arising nontransforming virus to the patterns of transforming virus is difficult to assess, the fraction of nontrans-
HANAFUSA
forming virus in the SR-RSV preparations used in this study was 25 to 33 %. Therefore, the patterns presented do primarily reflect the heat-dissociated RNA of transforming virus rather than nontransforming virus. However, we were unable to distinguish class a and b subunits as clearly as Duesberg and Vogt (1970) and MarOin and Ducsbcrg (1972). With SR-RSV D, the RKA peak containing the highest radioactivit’y could conceivably be considered as subunit b, with the heavier material comprising subunit a. But wit8h SR-RSV A and B-RSV, we were unable to different’iate subunits a and b. Although l\,Iartin and Duesbrrg (1972) also failed to demonstrate subunit a in B-RSV, they attributed their failure to t,he presence of excessive leukosis virus associated wit’h their B-RSV. In t,his study, however, the B-RSV preparations contained no or very minute amounts (< 0.1%) of leukosis virus and the specific activity of the polymcrase per transforming unit for B-RSV(f) suggests that the B-RSV preparations do not appear to contain “defective,” nontransforming virus in excess of t’ransforming virus. The development of a more refined technique with higher resolving power will hopefully provide better characterization of heat-dissociated RNA components. The expression of chf in C/E(+) cells had no detectable effect on the pattern of RAV-2 heat-dissociated RNA. But t’he major RNA species of B-RSV(f) grown in C/E(+) cells was consistently broader and more complex than that of B-RSV(-) grown in C/E( -) cells. One possibility that could account for the slight difference observed would be that viral specific RNA present in normal C/E(+) cells may contribute to the B-RSV(f) pattern. Although RAV-60 itself is biologically detected only in small amounts (T. Hanafusa et al., 1970a), the RNA of chf, the precursor of RAV-60, could be present within C/E(+) cells. Thus the intracellular pool of viral RNA in C/E(+) cells may be comprised of both B-RSV and chf elements. ACKNOWLEDGMENT The authors wish to thank Dr. Teruko Hanafusa for her contribution to some experiments and
RNA
OF AVIAN
Susan McRimmon, Lucy IXMauro, and Roni L. Bisdorfer for excellent technical assistance. This work was supported by U. 8. Public Health Service Research Grants CA-08747 and CA-12177 from the National Cancer Institute. CMS was supported by Damon Runyon Cancer Research Fellowship DRF-575-AT. REFISRRNCXS B.~nmr, J. P. (1970). Synthesis of the l<.NA of RNA-containing tumor viruses. Virology 49, 494-504. B.rumt, J. I’., and STIXE~? T. L. (1969). Analysis of the ribonucleic acid of murine leukemia virus. .I. C’irol. 4, 454-459. UISHOP, l>.H.L., CL.~YUROOI~, J. R..,and SPIEGELM.~N, S. (1967). Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Uol. Biol. 26, 373-387. and GRAE‘, T. (1971). Size IhLOON~XI, n. I’., differences among the high molecular weight RNA’s of avian tumor viruses. Virology 43,214222. Be.\y, (:. A. (1960). A simple efficient liquid scintillat,or for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. I, 279-285. CHAWFORD, L. V., AND CRAWFORD, E. M. (1961). The properties of Rous sarcoma virus purified by density gradient centrifugation. Virology 23, 227-232. I)UI<;SBRRG, P. II. (1968a). The RNA’s of influenza virus. Proc. Xat. Acad. Sci. U.S. 59, 930-937. ~)~I~:SUEHG, P. H. (1968b). Physical properties of ltous sarcoma virus RNA. Proc. 1\Tat. Acad. Sci. rr.,!%!60, 1511-1518. I)UBSHIZRG, P. II., and RUECKERT, It. R. (19G5). Preparative zone electrophoresis of proteins on polyacrylamide gels in 8 IIf urea. Anal. Biochem. 11, 342381. DUESBERG, P. H., and VOGT, P. K. (1970). Differences between the ribonucleic acids of transforming and non-transforming avian tumor viruses. Proc. -Vat. Acad. Sci. U.S. Gi, 1673lG80. ERIKSON, II. L. (1969). Studies on the RNA from avian myeloblastosis virus. Virology 3i, 124-131. FII:LDS, 1%.N., and JOKLX, W. K. (1969). Isolat,ion and preliminary genetic and biochemical characterization of temperature-sensitive mutan& of reovirus. virology 37. 335342. GOULD, H. J., PINDER, J. C., MATTHE:\VS, H. It., and GORDON, A. H. (1969). Fractionation of low molecular weight fragments of ribosomal and viral RNA by polyacrylamide gel electrophoresis. Anal. Biochem. 29, 1-21. HANAFUSA, H. (1965). Analysis of the defective-
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and a DNA endonuclease in virions of Rous sarcoma virus. Nature (London) 228, 424427. MONTAQNIER, L., GOLDI%, A., and VIGIER, P. (1969). A possible subunit structure of Rous sarcoma virus RNA. J. Gen. FiroZ. 4,449452. PARISH, J. H., and KIRBY, K. S. (1967). An extension of the naphthalene disulphonate method for mammalian nucleic acids. Biochim. Biophys. Acta 142, 273-275. PEACOCK, A. C., and DINGMAN,
C. W. (1968). Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agaroseacrylamide composite gels. Biochemislry 7, 66%
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amide gel electrophoresis of influenza virus RNA. Virology 34, 385-388. QUIQLEY, J. P., RIFKIN, D. B., and REICH, E. (1971). Phospholipid composition of Rous sarcoma virus, host cell membranes and other enveloped RNA viruses. Virology 46, 106-116. ROBINSON, W. S., and BALUDA, 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.
ROBINSON, W. S., PITKANEN,
A., and RUBIN, H. (1965). The nucleic acid of the Bryan strain of Rous sarcoma virus: Purification of the virus and isolation of the nucleic acid. Proe. Nat. Acad. Sci. U.S. 54, 137-144. SCHEELE, C. M., and HANAFUSA, H. (1971). Proteins of helper-dependent RSV. Virology 45, 401410. SHATKIN, A. J., SIPE, J. D., and LOH, P. G. (1968). Separation of ten reovirus genomes segments by polyacrylamide gel electrophoresis. J. ViroE. 2, 986-998. TONEGAWA, S., WALTER, G., BERNARDINI, A., and DULBECCO, R. (1970). Transcription of the SV40
genome in transformed cells and during lytic infection. Cold Spring Harbor Synap. Quad. Biol. 35, 823-831. VOGT, P. K. (1967). A virus released by “non-producing” Rous sarcoma cells. Proc. Nat. Acad. sci. U.S. 58, 801-808. VOGT, P. K. (1971a). Spontaneous segregation of NT viruses from cloned sarcoma viruses. Virology 46, 939-946. VOGT, P. K. (1971b). Genetically stable reassort-
ment of markers during mixed infection with avian tumor viruses. Virology 46,947-952. WATSON, J. D. (1971). The structure and assembly of murine leukemia virus: Intracellular viral RNA. Virology 45, 586-597.