Vol. 29, No. 2

JouRNAL OF VIRoLOGY, Feb. 1979, p. 475-482 0022-538X/79/02-0475/08$02.00/0

Effect of 5-Methylcytidine on Virus Production in Avian Sarcoma Virus-Infected Chicken Embryo Cells RAMAREDDY V. GUNTAKA,* RICHARD A. KATZ, AMY J. WEINER, AND MARGARET MOSKOWITZ WIDMAN Department ofMicrobiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received for publication 16 August 1978

5-Methylcytidine (5mC) is a minor constituent of RNA in procaryotes as well as eucaryotes. The function of this modified nucleoside is not known. We studied the effect of this compound on virus production in avian sarcoma virus-infected chicken embryo fibroblasts. We found, surprisingly, that virus release into the medium was severely reduced in cultures treated with 5mC. In contrast to the effect of 5mC on virus release, intracellular levels of virus-specific RNA transcripts as well as the proteins were slightly elevated. Analysis of intracellular RNA transcripts on velocity gradients and virus-specific proteins in polyacrylamide gels did not reveal any qualitative differences in 5mC-treated cells compared to cytidine-treated cells. From these results we conclude that the effect of 5mC is probably at the level of virus maturation or packaging. A large number of modified nucleosides are present as minor components in the nucleic acids of a variety of organisms (7). The predominant form of modification is methylation, and a vast majority of methylations are in the ribose moiety of RNA (13, 15, 21). Although it has been known for a long time that the methyl groups are introduced on nascent transcripts or on completed transcripts before maturation (13, 15, 21), the function of these methylated nucleosides is largely unknown. 5-Methylcytidine (5mO) is one such modified base which is common in all RNA species of eucaryotic as well as procaryotic cells so far studied (9). Approximately one to two residues of 5mC occur per 1,000 residues of RNA (3, 15). Four different mRNA's are shown to contain 5mC in varying amounts (2, 17, 18). In Sindbis viral 26S mRNA nearly 100% of the internal modification is present as 5mC (2). In an effort to study the role of modified nucleosides in transcription and processing of RNA, we have conducted a series of experiments in avian sarcoma virus (ASV)-transformed chicken embryo fibroblast cells. We have reasoned that if posttranscriptional methylation is required for processing of the precursor RNAs, then forced incorporation of modified nucleosides into RNA by exogenous addition might alter this process. This may result in the accumulation or enhanced degradation of precursor molecules. This approach might then be used to study the regulation of transcription and processing of ASV RNA. Studies initiated in our laboratory using

a cytidine analog, 5mC, show some dramatic and unanticipated results. The results presented in this paper will indicate that substitution of 5methyl-CMP for CMP in viral RNA suppresses virus production while allowing normal levels of transcription and translation of viral RNA. MATERIALS AND METHODS Cells and viruses. Secondary cultures of chicken embryo fibroblasts, prepared from 12-day-old embryos (SPAFAS, Conn.), were infected with ASV, Prague C, at low multiplicity. In about 10 to 12 days, the cells

were transformed as observed by their round morphology. The stock of virus used in these infections contained about 20 to 25% sarcoma virus, and the remaining fraction constituted transformation-defective virus as assayed by complementary (c) DNA4rV probe (19). Some experiments were done vith Prague B or B77td-infected cells with similar results. Virus stocks were propagated in chick cell monolayers in medium 199 supplemented with 10% tryptose phosphate broth, 0.2% sodium bicarbonate, 5% calf serum, and 1% dimethyl sulfoxide (20). Sometimes, when [3S]methionine- or 3P-labeling was done, Dulbeccomodified minimum essential medium with depleted amino acids and phosphate was used. In all labeling experiments, usually 3 to 5% dialyzed calf serum was used. Banding of ASV. The medium containing released virus was first centrifuged at low speed to remove cellular debris and cells, layered onto 8- to 9-ml 15 to 50% sucrose (0.06 M NaCl, 0.05 M Tris-hydrochloride, pH 8.1) gradients, and centrifuged in an SW41 rotor for 16 to 20 h at 30,000 rpm and 40C (5). Fractions were collected from the bottom of the gradient, and trichloroacetic acid-insoluble radioactivity was determined for each fraction. 475

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Isolation ofRNA. Total cellular RNA was isolated that pretreatment with preimmune serum greatly reessentially as described in an earlier report (6). This duced nonspecific binding of labeled antigens. The procedure was adopted from Glisin et al. (4). Briefly, immune complexes were washed three times with the celis were collected and lysed with 1% sodium phosphate-buffered saline containing 0.5% Nonidet Psarkosylate. After homogenization, 1 g of solid CsCl 40, 0.5% deoxycholate, and 0.1% gelatin. The radiolaper ml was added and then layered on a cushion of beled antigens were released from immune complexes the CsCl solution (1.35 g/ml). The cushion and its by boiling the pelleted bacterial cells in the 100-pl gel exact volume depended on the rotor used, but it was sample buffer (3% sodium dodecyl sulfate, 50 mM usually 2 to 3 ml. The samples were centrifuged in an Tris-hydrochloride, pH 6.8, 1% ,B-mercaptoethanol, SW50.1 or an SW50 rotor at 200C and 30,000 rpm for 10% glycerol, 0.001% bromophenol blue) for 3 min. 16 to 18 h. The pelleted RNAs were then taken up in The bacteria were removed by centrifugation, and the supernatants were electrophoresed on 10% polyacrylwater and used for further analysis. Preparation of 3H-labeled cDNA and nucleic amide gels, using the buffer system described by Laacid hybridizations. 3H-labeled cDNA was synthe- emmli (10) as modified by Blattler et al. (1). Gels were sized in an endogenous reaction with purified Prague fluorographed as described by Laskey and Mills (11). C virus, using high precursor concentrations (16). RaRESULTS dioactive deoxynucleoside triphosphate was present at 0.05 mM. Total DNA, synthesized under these condiIn the experiments described below, chicken tions, was sedimented in alkali sucrose gradients, and embryo cells were infected by a stock of ASV 3H-labeled DNAs larger than 14S and between 10 to which Prague C, contained mainly (70 to 80%) 14S were selected and used for hybridization. After self-annealing, these cDNA preparations contained transformation-defective particles. As a result, less than 3 to 4% double-stranded DNA as assayed by only a minor fraction (about 10 to 20%) of cells appear to be transforned as assessed by the Si nuclease (12). Hybridizations were carried out essentially as de- appearance of round cells. Addition of 1 mM scribed before (6). Approximately 1,000 cpm (20 x 106 cytidine did not appear to change this morpholcpm/,ug of DNA) were incubated in a 40-pl volume of ogy, but treatment with 1 mM 5mC for more 0.6 M NaCl-10 mM Tris-hydrochloride (pH 8.1)-3 than 24 h resulted in a dramatic enhancement mM EDTA with varying concentrations of ASV-inthe frequency (50 to 60%) of visibly round and fected cell RNA. The reactions were carried out for 15 in refractile cells. The packed cell volume also into 20 h. The extent of hybridization was monitored by creased in 5mC-treated cultures. Using trypan using S1 nuclease (12). When gradient fractions were assayed, RNA from blue exclusion to study viability, we ruled out each fraction was precipitated directly with ethanol in the possibility of cell death in 5mC-treated culFalcon plastic tubes (10 by 75 mm). After collecting tures, because there was no difference in the precipitates by centrifugation, a 40-p reaction mixture number of dead cells (less than 5%) in either containing approximately 1,000 cpm of 3H-labeled case. More rigorous experiments to correlate this cDNA was added, and hybridizations were carried out morphological change to actual transformation as above. Immunoprecipitation of viral proteins and have to be carried out to arrive at definitive polyacrylamide gel electrophoresis. ASV Prague conclusions. This preliminary observation prompted us to C, purified by banding in sucrose gradients, was disrupted by 0.5% Triton X-100 and mixed with Freund investigate further the effect of 5mC on viruscomplete adjuvant, and approximately 1 mg was in- specific RNA and protein levels. Before carrying jected intramuscularly into New Zealand white rab- out these experiments we had performed a numbits. The rabbits were boosted weekly with 500 jig of ber of experiments to study the rate and extent disrupted virus for 3 weeks and bled 6 days after the of cellular RNA synthesis. At various periods last boost. after treatment of transformed cells cytiASV-transformed chicken embryo cells, treated dine or 5mC, [3H]adenosine was addedwith for either with cytidine or 5mC, were labeled with [3S]methio- 30 min or 2 and the amount of label h, incorponine (Amersham Corp., >500 Ci/mol). Cell lysates were prepared and reacted with antivirus antiserum, rated into the total RNA was determined. The using Staphylococcus aureus as immunoabsorbent (8), data presented in Fig. 1 show that the rate of essentially as described by Opperman et al. (14). incorporation of [3H]adenosine into the total Briefly, this procedure consisted of the following steps: RNA was reduced to about 40% in 6 h. This 20 pl of preimmune serum was added to 0.5-ml cyto- decreased level of synthesis continued for at plasmic extracts and incubated for 15 min at 4°C, and least 3 to 4 days. Since pulse-labeling for short then 200 pl of a 10% Formnalin-fixed staphylococcus periods or long-tern labeling gave similar recell suspension (IgG-sorb, New England Enzyme Cenwe conclude that the effect of 5mC might ter) was added. After incubating at 4°C for 15 min, sults, bacterial cells were removed by centrifugation, and to be on the transcription of RNA rather than on the resulting supernatant 3 to 9 p1 of immune serum other aspects of RNA metabolism. Similar rewas added. After 30 min at 4°C, 200 A1 of a 10% duction in RNA synthesis was observed when staphylococcus suspension was added, and the incu- 'Pi was used instead of [3H]adenosine, suggestbation was continued for 30 min. Our experience was ing that the effect of 5mC was not on the uptake

5-METHYLCYTIDINE AND VIRUS PRODUCTION

VOL. 29, 1979 0

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FIG. 1. Effect of5mC on total cellular RNA, intracellular viral RNA, and virus production. ASV-transformed chick cells were treated with 1 mM cytidine or 5mC. At the indicated periods, one plate from each was labeled with [3H]adenosine (20 ,iCi/ml; specific activity, 37 Ci/mmol) for 2 h. The medium was collected, clarified by centrifugation, and banded in 15 to 50% sucrose gradients. The radioactivity under the peak was determined. The amount of radioactivity incorporated into cellular RNA was determined by measuring trichloroacetic acid-insoluble counts or from RNA isolated by the CsCl method (see text). Intracellular virus-specific RNA was assayed by nucleic acid hybridization with virus-specific 3H-labeled cDNA synthesized in an endogenous reaction. Each point was obtained from the Crti12 values of cytidineand 5mC-treated cellular RNA as described in the legend to Fig. 3. Symbols: 0, virus production; A, total cellular RNA synthesis; 0, intracellular virusspecific RNA.

of adenosine. Further studies strongly suggested that rRNA synthesis was preferentially suppressed by 5mC, although its incorporation could be demonstrated in both polyadenylic acid-containing [poly(A+)] and poly(A-) RNA (Guntaka et al., manuscript in preparation). Effect of 5mC on virus production. Since the primary effect of 5mC appears to be on RNA synthesis, we tested the effect of 5mC on virus production in cells infected by the Prague C strain of ASV. To do this, first we treated transformed cells with cytidine or 5mC. At different times, the cells were labeled with [3H]adenosine for 2 to 4 h, and then the media were centrifuged in 15 to 50% equilibrium density sucrose gradients. The results of each such experiment are shown in Fig. 2. ASV, released into the medium, banded at a buoyant density of 1.18 g/ml, as expected for type C particles. It is clear from the

477

data that in 5mC-treated cultures, virus release, as monitored by radioactivity at a density of 1.18 g/ml, was reduced by a factor of 7 to 8 (Fig. 2A). This result clearly shows inhibition of virus production by 5mC. It is possible that these results can be accounted for if the uptake of [3H]adenosine was inhibited by 5mC. However, the fact that similar data were obtained by monitoring virus production by either [3H]leucine (Fig. 2D) or 32Pi (Fig. 2E) argues against this possibility. That treatment with 5mC did not cause accumulation of particles devoid of nucleic acids was shown by labeling with [3H]leucine (Fig. 2D). Since there was no other peak of radioactivity at a lighter density, we conclude that the observed reduction of virus yields, in cultures treated with 5mC, was not due to the appearance of empty virion particles. Similar inhibition was observed in Prague B-transformed (Fig. 2B) or transformation-defective, virus-infected chicken embryo cells (Fig. 20). The virus production as assayed by focus formation or by measuring the newly synthesized DNA in infected quail tumor cells was also reduced by more than 10-fold (data not shown). Analysis of virus-specific RNAs and proteins. Since 5mC inhibits virus release into the growth medium, we asked whether virus-specific RNA was affected in any quantitative or qualitative way. This was checked by isolating total cellular RNA and hybridizing to 3H-labeled virus-specific cDNA. Total cellular RNA, from ASV-transformed cells treated for 24 h with cytidine or 5mC, was isolated by the CsCl method and hybridized to cDNA. The extent of hybridization was determined by using singlestrand-specific Si nuclease. The data presented in Fig. 3 indicate that in contrast to virus yields, intracellular virus-specific RNA levels were in fact unchanged or slightly elevated in 5mCtreated cells. Several different experiments clearly showed that in 5mC-treated cultures, a 50 to 100% increase over control values, as reflected by a decrease in Crtl/2, of about 20 was obtained with untreated or cytidine-treated RNA, indicating that approximately 0.1 to 0.15% of total cellular RNA is virus specific. Upon treatment with 5mC, however, a Crtl/2 between 8 and 12 was obtained, indicating that 0.18 to 0.25% is virus-specific RNA. It should be pointed out that the effect of 5mC was slightly better in Dulbecco-modified miniimal essential medium than in medium 199, probably due to the absence or reduced concentrations of nucleosides in the former. The overall effect of 5mC remained the same in either medium. These results were taken to mean that treatment of cells with 5mC resulted in virtually unchanged or increased levels of steady-state viral RNA. This could be due to

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FRACTION NUMBER FIG. 2. Banding of ASV in sucrose gradients. ASV-transformed cells were treated with 1 mM cytidine or 5mC for 24 h and then labeled with 10 ,uCi of [3H]adenosine per ml (specific activity, 37 Ci/mmol), 20 ,uCi of [3H]leucine per ml (specific activity, 61 Ci/mmol), or 20 ,uCi of carrier-free 32P-labeledphosphoric acidper ml (specific activity, 13 Ci/mmol) for 4 h. Media were collected, centrifuged for 10 min at 8,000 x g, and banded in 15 to 50% sucrose gradients as described in the text. Fractions were collected from the bottom of the gradients, and trichloroacetic acid-insoluble radioactivity was determined. (A) ASV Prague C (subgroup C) virus-transformed cells; (B) ASV Prague B (subgroup B) virus-transformed cells; (C) ASV B77td (subgroup C)-infected chick cells; (D) [3HJleucine labeling in ASV Pr-C-transformed cells; (E) 32P incorporation into ASV Pr-C-transformed cells. Symbols: 0, 1 mM cytidine; A, 1 mM 5mC; , no addition. 100

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FIG. 3. Effect of 5mC on intracellular viral RNA. ASV-transformed chick cells were treated for 24 h with 1 mM cytidine or 1 mM 5mC. Total cellular RNA was extracted by the CsCl method as described in the text. RNAs were dissolved in sterile distilled water, threefold dilutions were made, and appropriate volumes were hybridized with 3H-labeled cDNA (approximately 1,000 cpm; 40 Al in 0.6 M NaCI, 10 mM Tris-hydrochloride, pH 8.1, 3 mM EDTA) for 16 to 20 h. C,t values were computed from RNA concentrations. The percent hybridization was monitored by using Si nuclease. 3H-labeled cDNA, prepared as described in the text, reacted to more than 90%, with very little background (less than 4 to 5%). Symbols: 0, control RNA; A, 1 mM cytidine; 0, 1 mM 5mC.

enhanced rate of transcription or stabilization or preexisting viral RNA. Pulse-chase experiments should indicate either of these possibilities. It should be mentioned that the same results were obtained with cells treated with 5mC for up to 72 h (Fig. 1). Since virus-specific RNA levels appear to be unaffected by 5mC, we then performed experiments to determine whether there were any qualitative changes in various virus-specific mRNA's. Total RNAs were extracted from cells treated with cytidine or 5mC for 24 to 48 h and sedimented in velocity sucrose gradients. Fractions were collected from the bottom of gradients through a flow monitor, and portions from each fraction were hybridized with viral cDNA. The presence of 28 and 18S rRNA served as an intemal marker. A hybridization profile of the gradients identified two distinct size classes of virus-specific RNAs sedimenting as 35S and 24 to 28S (Fig. 4). Treatment of cells with 5mC did not reveal any difference among these two size classes. In agreement with the hybridization data (Fig. 3), 5mC enhanced the levels of all virus-specific RNAs as reflected by increased hybridization across the whole gradient. Therefore, from these experiments we conclude that absence of any particular virus-speciflc RNA is not the cause for decreased virus yields.

5-METHYLCYTIDINE AND VIRUS PRODUCTION

VOL. 29, 1979

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FIG. 4. Analysis ofASVRNA in sucrosegradients. ASV-transformed chicken embryo cells were treated with 1 mM cytidine or 5mC for 24 h. Cellular RNAs, extracted by the CsCl method, were dissolved in water, and approximately 50 pg for each sample, heated to 70°C for 3 min, was layered on 15 to 30% sucrose gradients. Sucrose solutions were made in 0.01 M Tris-hydrochloride-0.1 M LiCI-0.1% sodium dodecyl sulfate. Centrifugation was in an SW41 rotor at 20°C for 16 h at 24,000 rpm. Fractions were collected from the bottom of the gradient and were drawn through a flow monitor of a Gilford spectrophotometer to record the absorbance (optical density at 260 nm) directly. Approximately 0.4-ml fractions were collected. Samples, 0.1 ml, from each fraction were precipitated with ethanol, centrifuged, and assayed with 3H-labeled cDNA (860 cpm/fraction; specific activity, 20 x 106 cpm/pg) in a 40-,il reaction in 0.6 M NaCI-10 mM Tris-hydrochloride-3 mM EDTA at 68°C for 4 h. Each sample was then treated with 200 to 300 U of Si enzyme for 2 h at 50°C, and the Si-resistant fraction was determined. Symbols: 0, 1 mM cytidine; 0, 1 mM 5mC. Dotted line represents the tracing of absorbance at 260 nm.

Since we could not detect any major differin virus-specific RNA species, it was logical to study viral protein synthesis to understand the effect of 5mC on virus production. Before carrying out experiments on viral protein synthesis, we asked whether 5mC grossly damages cell protein synthesis machinery, since rRNA synthesis appears to be affected the most. Therefore, cells were labeled with [35S]methionine, and total cytoplasmic proteins were analyzed on polyacrylamide. It is clear from Fig. 5a that within the sensitivity of detection the pattern of protein synthesis appeared to be the same in cytidine- as in 5mC-treated cells. We then proceeded to identify virus-specific proteins by immunoprecipitating 35S-labeled proteins with rabbit anti-Prague C virus antiserum. Addition of increased amounts of antiserum to precipitate ences

479

viral antigens indicated that approximately 0.8 to 1% of the total cellular protein was virus specific. In all the experiments we consistently observed the same or slightly increased levels of viral antigens in 5mC-treated cultures (Table 1). Analysis of immunoprecipitates on polyacrylamide gel electrophoresis clearly showed that virtually all viral structural proteins were present in elevated quantities in 5mC-treated cells (Fig. 5b). The presence of Pr76, as well as p27, clearly suggests that 5mC does not interfere with processing. Pulse-labeling for 30 min in cells treated with 5mC for 48 h (Fig. 5b) also indicated that the rate of synthesis of viral proteins was not in any way affected by 5mC. However, processing of Pr76 to the final products appears to be somewhat slower than in the control (Fig. 5b, track I). The results (Table 2), therefore, provide strong evidence for a normal pattern of transcription and translation in 5mC-treated cultures. Yet, production of virus was substantially reduced, suggesting that the defect might be at the assembly or packaging steps. DISCUSSION The results presented in this report clearly indicate that 5mC, an analog of cytidine, can cause profound effects upon virus release. Since the same inhibition of virus production was observed by radiolabeling virus with [3H]adenosine, [3H]leucine, or 32Pi, it is very unlikely that the effect of 5mC is at the uptake of any nucleic acid precursor. Furthermore, since the levels of virus-specific RNA as well as viral proteins are slightly elevated in 5mC- compared with cytidine-treated cells, it is unlikely that the block of virus yields is at steps preceding transcription and translation. Thus, this is the first documented evidence for a nucleic acid analog affecting a critical function subsequent to transcription and translation of viral RNA. The maintenance of the same or elevated levels of viral RNA might be due to an increased rate of transcription or stabilization or preexisting RNA. This can be tested by pulsechase experiments, which are in progress. Preliminary results on the synthesis of total cellular RNA strongly suggest that poly(A+) RNA levels are, in fact, increased by about 50%, whereas poly(A-) RNA (presumably rRNA) is decreased (Guntaka et al., in preparation). Several possible hypotheses can be advanced to explain this unique phenomenon: (i) that incorporation of 5mC into RNA renders it less suitable for viral proteins to bind during packaging; (ii) that an increased rate of processing of 35S RNA into mRNA's may deplete the pool of 35S RNA which otherwise would be available

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pig-_ FIG. 5. Effect of 5mC on protein synthesis. Transformed chick cells were treated with 1 mM cytidine or 5mC for 16 h, during which time [3S]methionine (760 Ci/mmol) was added to the cultures continuously (a). Cytoplasmic extracts were prepared and analyzed as described in the text. In (b), cells were treated for 44 h with nucleosides and then pulse-labeled for 30 min or pulse-labeled followed by a 2-h chase. Immunoprecipitates were prepared and analyzed on 10% polyacrylamide gels. The arrows on the side of each figure indicates the positions of markers. (a) Fluorographs of [3S]methionine-labeled total cellular proteins. Tracks A, B, and C are 9, 6, and 3 ,l1, respectively, of labeled proteins from 5mC-treated cells; tracks D, E, and F are corresponding amounts from cytidine-treated cells. (b) Analysis of virus-specific proteins. Track A: 3S-labeled p27, p19 from purified virus. Arrows on the left indicate /3-galactosidase (135K) and bovine serum albumin (68K) markers. Tracks B to D: Immune precipitates from 48-h cytidine-treated cell extracts pulse-labeled for 30 min. One milliliter of cell lysate was incubated with 5 pIl ofpreimmune serum (track B) or 5 (track C) or 10 (track D) Al of immune serum. Tracks E to G: Immune precipitates from 48-h 5mC-treated cell extracts pulselabeled for 30 min. One milliliter of cell lysate was incubated with 5 ,u ofpreimmune serum (track E) or 5 (track F) or 10 (track G) ,il of immune serum. Track H: Immune precipitates from 48-h cytidine-treated cell extracts pulse-labeled for 30 min with [3S]methionine followed by a 2-h chase with a 50-fold excess of unlabeled methionine. Track I: As for track H, except 5mC replaced cytidine. In tracks H and I, 10 [lI of immune serum was used.

5-METHYLCYTIDINE AND VIRUS PRODUCTION

VOL. 29, 1979

TABLE

1. Detection of virus-specific proteins by

immunoprecipitation

5mC

Cytidine

Amt of

Type of serum added

RadioacRiadioactivtivity im- % In- precipitated imnmunomunopre- put ity

cipitated

(cpm)

(cpm)

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PI)

Anti-Prague C antiserum (9

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pecific pro-

tern (% input)

12,540

0.13

15,510

0.15

48,180

0.5

57,420

0.54

77,550

0.81

94,710

0.90

I) 10.5 x 106

Total input 9.6 x 106 (cpm)

TABLE 2. Summary of the effect of 5mC on virusspecific RNA and virus production Rate Rate of of ceR celpro-

Virus.

Virusspe-

Ratio of viral yields RNA

Virus

specific cif rus retein thsynhesNA _ leased (%M M i (% 1 100 100 100 Cytidine 100 100 30-40 60-100 150-300 >100 15-20 10-15 5mC Treatment RNA

tern

481

interact with methylated RNA may be reduced. The evidence that all the structural viral proteins are synthesized normally argues against any errors in the processing as well as the translation of viral RNA transcript. Preliminary evidence indicates that 5-methylCMP incorporates equally well into poly(A+) and poly(A-) RNA, yet synthesis of poly(A-) RNA is preferentially inhibited (Guntaka et al., in preparation). Since the majority of poly(A-) RNA is ribosomal, this means that transcription of rRNA is blocked by 5mC. Whether this effect is on RNA polmerase I or a feedback inhibition due to the absence of rRNA maturation remains to be studied. Continued studies with 5mC may reveal some subtle stages in the mechanism of virus packaging and probably ribosome formation. ACKNOWLEDGMENTS The technical assistance of Geryl Jackson and Lou Yesner is gratefully acknowledged. We thank H. S. Ginsberg and H. S. Y. Young for the critical reading of the manuscript. This work was supported by Public Health Service grant CA 19152 from the National Cancer Institute.

LITERATURE CITED 1. Blattler, D. P., F. Garner, K. Van Slyke, and A. Bradley. 1972. Quantitative electrophoresis in polyacrylamide gels of 2 40%. J. Chromatogr. 64:147-155. 2. Dubin, D. T., and V. Stollar. 1975. Methylation of sindbis virus 26S messenger RNA. Biochem. Biophys. Res. Commun. 66:1373-1379. 3. Dunn, D. B. 1960. The isolation of 5-methylcytidine from RNA. Biochim. Biophys. Acta 38:176-178. 4. Glison, V., R. Crkenjakov, and C. Cyirs. 1974. Ribo-

for virus formation; and (iii) that cellular rRNA nucleic acid isolation by Cesium chloride centrifugation. is required for virus production and, therefore, Biochemistry 13:2633-2637. inhibition of rRNA synthesis reduces virus 5. Guntaka, R. V., B. W. J. Mahy, J. M. Bishop, and H. yields. E. Varmus. 1975. Ethidium bromide inhibits the apThe data presented in Fig. 4 strongly suggest pearance of closed circular viral DNA and integration of virus-specific DNA in duck cells infected by avian that at least there are no recognizable differences virus. Nature (London) 253:507-511. in processing since there is no evidence of accu- 6. sarcoma R. V., and A. J. Weiner. 1978. Effect of Bt2 Guntaka, mulation of 35S RNA. Moreover, in agreement cAMP on the intracellular levels of avian sarcoma virus with hybridization data in Fig. 3, all the species specific RNA. Nature (London) 274:274-276. of viral RNA appear to be enhanced to the same 7. Hall, R. H. 1971. The modified nucleosides in nucleic acids. Columbia University Press, New York. level. Therefore, it is very unlikely that an in- 8. Kessler, S. W. 1975. Rapid isolation of antigens from celLs creased rate of processing of 35S RNA is responwith a staphylococcal protein A antibody absorbent: sible for the reduced yields of virus. However, parameters of the interaction of antibody-antigen complexes with protein A. J. Immunol. 115:1617-1624. from these data it is very difficult to distinguish 9. Klagsbrun, M. 1973. An evolutionary study of the methbetween alternatives (i) and (iii). ylation of transfer and ribosomal RNA in prokaryPulse-chase experiments to demonstrate proote and eukaryote organisms. J. Mol. Chem. 248: the rate and that tein synthesis strongly suggest 2512-2520. extent of viral protein synthesis are unaffected 10. Laemmli, V. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. by 5mC, which also indicates that the effect of Nature (London) 227:680-685. 5mC is at a step subsequent to protein synthesis. 11. Laskey, R. A., and A. D. Mills. 1975. Quantitative film Since it has been shown that Pr76 is processed detection of 3H and "4C in polyacrylamide gels by fluoin the immature virions (22), it is possible that rography. Eur. J. Biochem. 56:335-341. L. Fanshier, the reduced rate of processing of Pr76 to its final 12. Leong, J. A., A. Garapin, N. Jackson, W. Levinson, and J. M. Bishop. 1972. Virus-specific products might account for the diminished yield ribonucleic acid in cells producing Rous sarcoma virus: of the virus. If so, this is in accordance with our * detection and characterization. J. Virol. 9:891-902. of Pr76 to 13. Maden, B. E. H., and M. Salim. 1974. The methylated current hypothesis that the affinity

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15. 16.

17. 18.

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