The genesis of rous sarcoma virus messenger RNAs

VIROLOCY112,714-728 (1981)

The Genesis of Rous Sarcoma

Virus Messenger

RNAs

PERRY B. HACKETT, HAROLD E. VARMUS, AND J. MICHAEL Department of Microbidogy

and Immunology, Unive-rsit~ of Califwnia,

BISHOP’

San Francisco, Ca&fbmia @I@

Accepted February 11, 1981 Cells infected with Rous sarcoma virus (RSV) produce three classes of virus-specific mRNAs-one that has the size of the viral genome and two that are smaller than the genome. We have explored the mechanisms by which these viral mRNAs are produced. The site for initiation of viral RNA synthesis was located by using irradiation with ultraviolet light to inactivate transcription in infected cells. The metabolism of viral RNA was elucidated by the use of glucosamine to effect satisfactory pulse-chase experiments. We conclude that transcription from the RSV provirus initiates at or near the nucleotide that encodes the 5’ terminus of the viral genome. The primary transcript is subsequently processed to generate the subgenomic mRNAs, which appear independently of each other between 15 and 45 min after the synthesis of their precursor. Viral RNA has several distinctive fates: ca. 25% remains in the cell in the form of the genomic length mRNA; lo-20% takes the form fo subgenomic mRNAs; and the remaining 56% disappears from the cell, presumably because of export as viral genome (but perhaps also as a consequence of degradation within the cell). Our data provide a functional definition for the initiation of viral RNA synthesis and conform to the results of recent structural studies that have identified a possible “promoter site” near the ends of the RSV provirus.

gene product-the glycoprotein of the viral envelope (Pawson et al., 1977). Rous sarcoma virus (RSV) is a RNA tu(iii) mRNA” (A& 1.1 X 106) encodes the mor virus with a diploid, single-stranded 3’ terminal erc gene; the product of this RNA genome of approximately 9000 nu- gene is responsible for neoplastic transcleotides (Bishop, 1978). The integrated formation of infected cells (Beemon and DNA copy (or provirus) of this positiveHunter, 1977; Kamine et al., 1978). stranded genome serves as template in the All three mRNA species have: (1) a 5’ production of three classes of viral mes- terminal “cap” (Mellon and Duesberg, senger RNAs (Hayward, 1977; Weiss et al., 1977), (2) an identical leader sequence at 1977; Lee et al., 1979; Quintrell et cd.,1980). their 5’ ends (Cordell et al., 1978), and (3) (i) rnRNAO@& (Mr 3.3 X 106) contains polyadenylated 3’ termini (Bender and all four of the RSV genes and comprises Davidson, 1976; King and Wells, 1976). messages for both the gag gene products Viral RNA is synthesized in the nuclei of (the structural proteins of the viral core infected cells by host RNA polymerase II (von der Helm and Duesberg, 1975, Paw- (Jacquet et al., 1974; Rymo et al., 1974), but son, et al., 1976; Pawson et al., 1977)), and the sites at which viral RNA synthesis inithe pal gene product (viral reverse tran- tiates on the DNA template have not been scriptase (Paterson et al., 1977; Purchio et rigorously identified. According to recent al., 1977; Weiss et al., 1978)). results from sequencing of cloned viral (ii) mRNA” (Mr 1.8 X 106) encodes the DNA (Czernilofsky et al., 1980), retrovirus two genes in the right-hand half of the proviruses may carry promoters that are provirus and is the messenger for the env located so as to permit initiation of transcription at the 5’ end of the viral genome. Moreover, precursor RNAs larger than the 1To whom reprint requests should be addressed. INTRODUCTION

0042-6822/81/166714-15$02.66/O Cqyrigbt 0 1981 by Academic Press, Inc. All rigbta of reproduction in any form reserved.

714

GENESIS

OF ROUS SARCOMA

haploid subunit of the viral genome have not been identified (although there have been tentative suggestions that a small fraction of virus-specific RNA in the nucleus may be larger that the haploid subunit of the genome (Haseltine and Baltimore, 19’76; Fan, 1977)). None of the available data provide a functional definition of promoters for retrovirus RNA synthesis, however, and little is known of the mechanisms the generate the subgenomic viral mRNA. We have mapped the promoters for synthesis of RSV RNA in infected cells by using ultraviolet (uv) light to inactivate transcription in a dose-related manner (Sauerbier, 1977). This procedure has been used successfully to map the ribosomal RNA (rRNA) genes of eukaryotic cells (Hackett and Sauerbier, 1974), to locate the promoters for several structural genes in normal vertebrate cells (Giorno and Sauerbier, 1976; Goldberg et al., 1971; Gilmore-Hebert et al., 1978; Giorno and Sauerbier, 1978; Hackett et al., 1978), and to identify sites for initiation of transcription in the genomes of adenovirus (Girvitz and Rainbow, 1971; Berk and Sharp, 1977; Goldberg et al., 1971; Goldberg et al., 1978)), vesicular stomatitis virus (Ball and White, 1976; Ball, 1971; Colonno and Bannerjee, 1977), rabies virus (Flamand and Delagneau, 1978), and vaccinia virus (Pelham, 1977; Bossart et al., 1978). We also examined the kinetics of RSV mRNA formation by exploiting the fact that glucosamine can be used to effect satisfactory pulse-chase experiments with [SH]uridine in eukaryotic cells (Scholtissek, 1971; Wertz, 1975; Herman and Penman, 1977). Our results indicate that transcription from the ASV provirus initiates at or near the nucleotide representing the 5’ terminus of the viral genome, and that the subgenomic mRNAs are independently derived from an RNA that is similar or identical in size to the haploid subunit of the viral genome. MATERIALS

AND METHODS

Materials. Proteinase K was obtained from E. M. Laboratories, 3~ crystalline

VIRUS

MESSENGER

RNAs

715

trypsin from Worthington, methyl mercuric hydroxide from Alpha Division-Ventron Corporation, oligo( dT)-cellulose (type 3) from Collaborative Research Inc., and hydroxyapatite (Bio-Gel, HTP grade) from Bio-Rad Laboratories. CeUs and viruses Chicken embryo fibroblasts were transformed by a clone of the Prague B (Pr-B) strain of RSV provided by P. Vogt. The chick fibroblasts used in these studies were free of endogenous viral gene products (“chick helper factor” and “group-specific antigen”). Infected cells were used between the third and sixth passages, when their morphology indicated complete transformation. Concentrated Pr-C RSV, used to make viral cDNA, was obtained from University Laboratories through the auspices of the Office of Program Resources and Logistics, National Cancer Institute. D-Glucosamine, uridine, and cytidine, all A grade, were bought from Calbiochem. [SIJIJridine came from Amersham (30.1 Ci/mmol) or New England Nuclear Corporation (29.35 Ci/mmol), [q]orthophosphate came from ICN.

Prelabeling and ultraviolet irradiation Standard media (containing 1% dialyzed fetal calf serum) without nucleosides but with [‘Hluridine (106 &i/ml) was added to all plates 8-10 hr before ultraviolet light (uv) irradiation and the cells were incubated at 41’. The radioactive media was then removed and the plates washed twice with prewarmed media. The wash media was removed and the plates were individually irradiated, or mock irradiated, with a GE G2T2 low-pressure Hg lamp which emitted primarily at 254 nm. We used a dose rate of 26 ergs/mm2 . set as measured with a YSI-Kettering Model 65 Radiometer loaned to us by R. B. Painter. Total time for washing and irradiating the cells was approximately 3 min.

P&e tabelhg ad preparation of cellular lvsate& After uv irradiation, 4 ml pulse-labeling media containing 82p (2 mCi/ml) was added to plates which had been prelabeled with r)I)uridine. The pulse-labeling media contained 1% dialyzed fetal calf serum and lacked phos-

716

HACKETT,

VARMUS,

phate. The cells were labeled for 2 hr at 41” unless otherwise indicated. After pulse labeling, the total nucleic acid of each culture was isolated as described earlier (Weiss et al., 1977) with the following modifications: (1) 20 pg 3X crystallized trypsin was added to the Punk’s saline for removal of the cells from the culture plates; (2) proteinase K was used at a concentration of 500 rg/ml for digestion of cellular protein; (3) the lysates were deproteinized two times with cold phenol:chloroform:isoamyl alcohol (1:1:0.02). The nucleic acids were then precipitated by the addition of 2.5 vol of 95% ethanol and 0.1 vol 2 M NaAc. Fractionation of RNA. Poly (A)-containing mRNA was separated from other nucleic acids by oligo(dT)-cellulose chromatography (Weiss et CCL,1977). Viral mRNA was isolated by molecular hybridization to viral cDNA (Pr-C strain of RSV) made as described (Ringold et al., 1977; Cordell et aZ., 1978). For this procedure, the total nucleic acid from lo7 cells was resuspended in 30 ~15 mM EDTA to which 60 ~1 deionized formamide, 20 pg cDNA, and 12 gl 4 M phosphate buffer (pH 6.8) were added, the 122-11 reaction volumes were incubated 4-8 hr at 40”. The reaction mixes were diluted with 120 ~1 Hz0 plus 1 ml wash buffer (8 M urea, 0.2 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate) and then chromatographed over l-ml hydroxyapatite columns equilibrated in wash buffer at 41”. The columns were washed with 10 ml wash buffer; the viral RNAs and cellular DNA were eluted with 5 ml 0.4 M phosphate buffer (pH 6.8) at 50”. The eluted viral RNAs were dialyzed against 300 vol. of 300 mM NaCl, 10 m&I Tris (pH 7.6), 10 mMEDTA, 0.1% sodium dodecyl sulfate for at least 20 hr before precipitation with 2.5 vol of 95% ethanol. Cellular DNA coisolated with viral RNA by hydroxyapatite chromatography was enzymatically digested with iodoacetatetreated DNase at 40 &ml in 7.5 m&f MgClz, 10 mM Tris (pH 7.6) as described previously (Pelham, 1977; Cordell et al., 1978). Separation of RNAs by electrophoresis on denaturing agarose gels. The various preparations of RNA were resuspended in

AND BISHOP

30 ~1 0.5 X borate buffer (pH 8.1) (Bailey and Davidson, 1976) to which methyl mercuric hydroxide was added to 10 mM. The samples were electrophoresed on 1.2% agarose gels in borate buffer containing 5 miU methyl mercuric hydroxide. After electrophoresis, the gels were soaked for 30 min in a solution of 10 pg/ml ethidium bromide containing either 500 mM ammonium acetate or 20 m&Z2-mercaptoethanol. The gels were destained in water for 30 min. Gels containing rRNAs were divided into 2-mm (X4 X 14 mm) slices which were digested in 0.1 ml 60% perchloric acid. Tritosol was added to each sample and the radioactivity was measured in a liquid scintillation counter. Gels containing polyadenylated mRNA were either sliced into 2-mm slices as described above, or only the regions of the gel in which the three viral mRNA species migrated were excised. Analysis of RSV RNA. We have described the procedure in detail elsewhere (Cordell et al., 1978). Gel slices were dissolved in 8 M NaC104 (33 ~1 for each 100 ~1 of gel) at 68” for 20 min. Thereafter, 2 c(g RSV cDNA, 20 pg yeast and 0.4 ~1 2mercaptoethanol were added and the sample divided in half. Fifty micrograms of RSV RNA (obtained from concentrated Pr-C virus) was added to one-half to serve as competitor; an equivalent volume of 3 mM EDTA was added to the second half. The samples were incubated under mineral oil at 68” for between 48 and 72 hr, treated with 1 pg RNase A and 20 units RNase TI for 30 min at 37’, and then diluted into wash buffer for hydroxyapatite chromatography as described above. Fractions from the hydroxyapatite column were diluted into Hz0 and tritosol and the amounts of *P and 3H were measured in a liquid scintillation counter. The results obtained from counting RNA containing both =P and 3H were corrected for disintegrations of 3zPrecorded in the 3H channel; the percentage of 3H disintegrations recorded in the BP channel was less than 0.1%. Measurements carried out in the presence of the unlabeled RSV RNA provided an estimate of the background in the

GENESIS OF ROUS SARCOMA VIRUS MESSENGER RNAs

assay-ca. 0.02-O.lO%, as reported previously (Lee et aZ., 1979). RESULTS

Models of the RSV Transcriptional Unit Our goal was to determine the number and approximate location(s) of promoters for the RSV mRNAs. The alternatives are illustrated in Fig. 1. Model A describes the possibility of a single promoter located either within the leftward domain of the provirus (as illustrated) or in cellular DNA at some point to the left of the provirus. These two possibilities are fomally equivalent in our experiments, differing only in the apparent target size for inactivation of the template and in the extent to which processing is required to generate the mature viral geModels

of the ASV Transcriptional

I

8P-w) PI

L

A

A

P2

P3 w

m

I--

unit

mRNASrC mRNAe”” ,,,RNAga!#d

FIG. 1. Models of the RSV transcriptional unit. Model A: Single promoter model. The primary transcript is processed into the mature mRNA*‘&, mRNA”““, and mRNArr” molecules. Model B: Multiple promoter model. Transcription of rnRNAW’&. mRNA”“, and mRNAm are each initiated at separate promoters, designated PI, P2, and Pl, on the same provirus. Possible precursor transcripts are not shown. In both models, the integrated provirus (double lines) is shown to have the redundant sequences designated m, each mature RSV mRNA contains a copy of n at its 5’ end and a copy of q at its 3’ terminus. The terminal redundancies are not drawn to scale. The leader sequence on each RSV mRNA is larger than the the 101 nucleotide 5’ terminal redundancy designated by the closed box on each message. Our unpublished data indicate that the length of the leader is about 389 bases.

717

nome. The subgenomic mRNAs would arise from processing of the primary transcript, and transposition of the 5’ leader sequence would occur by splicing (Berget et al., 1977; Chou et al., 1977; Gelinas and Roberts, 1977; Klessig, 1977). Model B illustrates the possibility that separate promoters might give rise to each of the viral mRNAs. This scheme is formally equivalent to the now disproven possibility that each mRNA is transcribed from a separate and distinctive provirus (Quintrell et al., 1980). Moreover, it does not easily account for the splicing of a 5’ leader on to each mRNA. As in model A, the promoters could be located some distance to the left of the bodies of each of the mRNAs; this situation would be manifest in the target sizes of the templates. We used inactivation by uv irradiation to determine the number of promoters for the viral mRNAs and to distinguish between the two models. This procedure is based on the fundamental postulate that uv irradiation of DNA in vivo or in vitro results in the random formation of transcription-terminating lesions; at such sites both RNA polymerase and a truncated RNA transcript are released. Consequently, the rate of formation of a primary transcript from a specific transcriptional unit decreases exponentially with increasing uv dose, and the sensitivity of transcription to inactivation is proportional to the length of the DNA template. Thus, if model A were correct, exponential inactivation of synthesis with increasing uv dose should be same for all three mRNAs; whereas, if model B were correct, we would expect the inactivation of transcription of the genes coding for the three mRNAs to be in the approximate proportion of the molecular weights of the RNAs, 3.3, 1.8, and 1.1 X 106.

Experimental Strategg In order to determine the inactivation coefficients for the three viral mRNAs, we needed to measure the amounts of synthesis of each RNA species after uv irradiation and to distinguish this RNA from the bulk of the viral RNA which existed

‘718

HACKETT, VARMUS, AND BISHOP

in the cytoplasm of the infected cells prior to irradiation. Our procedure was to incubate several monolayers of infected cells for 8-10 hr with [3H]uridine (see Methods) to prelabel uniformly all species of RNA. After removing the radioactive media, washing the cells, and irradiating with uv light, the cells were pulse labeled for 2 hr with [32P]orthophosphate. By this procedure of double labeling, we could distinguish RNA made after uv irradiation (32P pulse-labeled RNA) from the preexisting RNA (3H prelabeled), and we could determine the efficiency or our extraction of RNA samples by reference to the amounts of the prelabeled RNAs obtained from the different cell lysates.

Ultraviolet-Induced Temination of Transcription of the rRNA Genes We first measured the rate at which transcription of the rRNA genes was reduced after irradiation with uv light. Since the size of the rRNA transcriptional unit is known, as are the relative locations of the 18 S and 28 S rRNA genes (Schibler et al., 1975), these measurements provided standards to which we could later compare the sensitivities of RSV genes. Double-labeled cellular RNA was electrophoresed in denaturing agarose gels, which were sliced to determine the amounts of =P and 3H for each rRNA. The mRNA that was eluted from the oligo(dT)-cellulose was used for analyzing transcription of the RSV genes after irradiation (see the following section). The decrease in rRNA synthesis after uv irradiation is evident from the data shown in Fig. 2. The amounts of [3H]uridine in the two major rRNA species (18 S and 28 S) were nearly the same in the four samples of RNA, whereas the relative amounts of 32P-labeled rRNA decreased with increasing uv doses. As expected from previous experiments in which the uv sensitivities of rRNA genes were examined (Hackett, 1974; Hackett and Sauerbier, 1974a; Hackett and Sauerbier, 1974b), transcription of the promoterproximal 18 S rRNA gene in avian cells was less sensitive to uv irradiation than the 28 S rRNA gene, which is at the pro-

0 I-----

415 28s 185 141

b 415 28s 4 1.

18s +

ii

P 0 c

d

; 4

I A I I

10

20

30

mm FROM ORIGIN FIG. 2. Fractionation of ribosomal RNAs by electrophoresis in agarose gels. Cultures of Pr-B virusinfected cells were prelabeled for 8 hr with [3H]uridine, irradiated with uv light for (a) 0, (b) 7, (c) 14, (d) 21 set, and pulse labeled for 2 hr with [?PJorthophosphate. Nonadenylated RNA was isolated and electrophoresed on denaturing agarose gels, which were sliced into 2-mm slices, dissolved in 0.1 ml 60% perchloric acid, and counted in a liquid scintillation counter. The dashed lines show the 3H cpm corrected for spillover of =P cpm. The points show the =P cpm.

moter-distal end of the rRNA transcriptional unit (Schibler et al., 1975). The dose-response curves for the inhibition of rRNA synthesis (Fig. 3) were derived from several experiments like that shown in Fig. 2. The inactivation coefficient for the 41 S precursor RNA (the initial transcript of the rRNA transcriptional unit (Attardi and Amaldi, 1970)) was the same as that for the 28 S rRNA gene, as expected since the 28 S gene is located at the far end of the transcriptional unit (Schibler et al., 1975). The rate of processing of 41 S and 32 S precursor

GENESIS OF ROUS SARCOMA VIRUS MESSENGER RNAs

I

I

I

719

transcriptional unit (approximately 12,000 base pairs). The inactivation coefficient (the reciprocal of the I& value) for 28 S and 41 S rRNA is therefore (13 set)-‘. The second notable aspect of the two curves is that they intercept the ordinate at about 125% rather than at 100%: this anomaly is discussed below and in the accompanying paper. Inhibition

of RSV rnRNA Synthesis by uv

We examined the inhibition of synthesis of each of the RSV messengers by analyzing the polyadenylated mRNA fractions from the oligo(dT)-cellulose chromatograSECONDS U.V. phies described above. Preparations of FIG. 3. Dose-response curves for rRNA synthesis mRNA were fractionated by electrophoafter irradiation of infected chick cells with uv light. resis in agarose gels. The relative amount Six experiments of the type shown in Fig. 2 were of virus-specific RNA in fractions of the conducted to determine the dose-response curves for gels was then determined by molecular 18 S, 23 S, and 41 S rRNA. The residual rate of rRNA hybridization with virus-specific cDNA synthesis for each species was calculated as previaccording to a previously described proously described (Hackett and Sauerbier, 1974b). l , cedure (Weiss et al., 1977); this assay has 18 S rRNA; 0,28 S (with 32 S) rRNA; n , 41 S rRNA. a background of approximately 0.02-0.10% The slopes of the two lines for 28 S and 18 S rRNA were determined by the method of least squares and with RNA from unifected cells (Dee et al., they intercept the ordinates at 12’7 and 122%, re- 1979, and unpublished data of the auspectively. The Dn for the dose-response curve is 27 thors). set; for the 23 S curve the On is 13.0 sec. The corFigure 4 illustrates the resolution we relational coefficient for the 18 S curve is 0.92 and achieved for virus-specific RNA and for for the 28 S curve is 0.97. cellular RNA (largely rRNA) that did not hybridize with virus-specific cDNA. The efficiency of hybridization was monitored rRNAs into mature 18 S and 28 S rRNA by the inclusion of a trace amount of %Pwas not inhibited to any measurable ex- labeled viral RNA in each assay; hytent by uv irradiation (Hackett, 1974; bridization of this tracer was uniform throughout the entire series of assays (as Hackett and Sauerbier, 1974a; Hackett and Sauerbier, 1974b; Hackett et aZ.,1981). denoted by the dashed line in Fig. 4). As Two aspects of the dose-response curves anticipated, three species of viral RNA are significant. First, the data for 28 S were identified, with molecular weights rRNA (and 41 S precursor rRNA) repre- corresponding to the values previously desent the sensitivity of a transcriptional termined for the three mRNAs of RSV unit that directs the synthesis of the 41 (Weiss et al., 1977; Lee et al., 1979). The S RNA whose mass is 3.9 X lo6 daltons peaks of subgenomic viral mRNAs rise 3( Attardi and Amaldi, 1970). The 28 S curve to lo-fold above the baseline value of hyis thus an internal standard which can be bridization. We attribute the baseline to used to calibrate the relative transcripdegradation products from rnRNA#&“l tional sensitivities of other genes. A dose and estimate that these products account of 260 ergs/mm’ (13 set) lowers the rate for ca. 30% of the hybridized RNA in the of 41 S rRNA synthesis to 3’7% and there- region of mRNAenV,ca. 20% in the region fore introduces an average of one tranof mRNA”. As explained below, contamscription-terminating lesion in each rRNA ination of this magnitude did not appre-

HACKETT, VARMUS, AND BISHOP

720

2800 ’ ii E 2

2400 2000 r .

SLICES(2 mm) FIG. 4. Fractionation of RSV-specific RNA by electrophoresis in an agarose gel. The polyadenylated RNA from a culture of cells labeled with rH]uridine for 8 hr was isolated and electrophoresed on 1.2% agarose gels. Sequential 2-mm slices were dissolved in 8 M NaClO’ and virus-specific RNA was detected by molecular hybridization, as described under Materials and Methods. @P-Labeled RSV virion RNA was included in each hybridization reaction as a standard for the efficiency of hybridization. The dashed line represents an extrapolation designed to show the approximate contribution of mRNAm’d fragments in the regions of the gel containing the subgenomic RSV mRNAs. (0) ‘H-Labeled virus-specific RNA; (0) ‘H-labeled cellular RNA unreactive with RSV cDNA; (- - -) percentage of =P-labeled RSV virion RNA hybridized to cDNA.

ciably distort the outcome of our uv mapping experiments. In order to quantitate the effect of ultraviolet irradiation on the synthesis of RSV mRNAs, we employed two radioactive labels, as described above for rRNA. Infected cells were labeled approximately to steady state with [3H]uridine, irradiated, and then pulse labeled with =P. Preparations of mRNA were extracted and purified following the pulse labeling and then fractionated by electrophoresis. The samples of RNA from irradiated cells

were analyzed in parallel with a preparation of =P-labeled RSV mRNAs that had been purified in advance by molecular hybridization (Weiss et al., also see Methods). The location of the purified viral mRNAs following electrophoresis could be determined by brief autoradiography. Corresponding regions of the lanes containing the irradiated samples were then excised and used for the measurement of virus-specific RNA by quantitative molecular hybridization. Representative results are presented in Table 1. (In preliminary experiments modeled after the protocol of Fig. 4, we determined that ultraviolet irradiation did not give rise to distinct new species of viral RNA.) Dose-response curves for the three RSV RNA species were determined by compiling the results of five such experiments; the curves are plotted in Fig. 5. Two features are evident in this figure. First, all three dose-response curves have nearly the same slope, indicating that the uv inactivation coefficients for transcription of the three major mRNAs were similar. The inactivation coefficients are (13.5 set)-’ for mRNAm’@, (12.0 set)-’ for mRNA”, and (12.5 set)-’ for mRNA”. Second, as with the rRNA dose-response curves, the RSV RNA curves have shoulders, and extrapolation of the exponential aspects of the curves resulted in ordinate intercepts between 120 and 130%. An analysis of the amounts of genome RNA in virus particles released after uv irradiation showed that the decrease in pulse-labeled, RSV RNA in virions was similar to that in the cells (unpublished data). Also shown in Fig. 5 are theoretical dose-response curves expected for the viral mRNAs if each had its own promoter, as modeled in Fig. 1B. Construction of these curves was based on the assumption that the rate of formation of transcription-terminating lesions on the RSV provirus is the same as on the rRNA cistrons (see below). For the theoretical calculation, the ordinate intercept was placed at 125%, since this was the approximate value for all of the RSV mRNA and chick rRNA dose-response curves. As discussed in the accompanying paper to this manu-

721

GENESIS OF ROUS SARCOMA VIRUS MESSENGER RNAs TABLE 1 MEASUREMENTOF VIRUS-SPECIFICRNA IN SAMPLESFROMACAROSEGELS” RNA mRNAL”“/h”

Dose 6434 0 7 14 21

mRNA’““

mRNA””

32p

32P/3H

622

1048

589

709 479 279

1.68 1.20 0.67 0.40

100 71 39

2.31

100

1.39 0.98

60 42 28

720

699

0 7

270

14 21

255 410

0 7

150

14 21

Percentage control

3H

490

294 205 320

625 680 250 270

0.66

190

2.93 1.94 0.93

235

0.73

440 560

24

100 65 32 25

O1 RSV-infected cells were labeled to approximately steady state with [3H]uridine, irradiated, and pulse labeled with =P. The mRNAs were fractionated by electrophoresis in an agarose gel. Virus-specific RNAs were located by reference to the positions of purified viral RNAs analyzed in a parallel lane of the gel (see text). Appropriate regions of the gel were excised, dissolved in 8 M sodium perchlorate, hybridized with RSV cDNA, and fractionated on hydroxyapatite, all as described under Materials and Methods. For each sample, a duplicate determination was performed in the presence of a relatively large quantity (50 pg) of unlabeled RSV RNA in order to obtain an operational value for the background hybridization (see Materials and Methods). The data are reported here as the difference between the numbers obtained in the absence and presence of unlabeled competitor RSV RNA. Backgrounds in the presence of competitor ranged from 40 to 60 cpm for all 32P-labeled samples; from 120 to 210 cpm for [3H]mRNA*“V’P”‘; from ‘70 to 150 cpm for [3H]mRNA”“; and from 30 to 90 cpm for [3H]mRNA”. The ratio of 3ZP/3H reflects the relative efficiency of mRNA biogenesis after, as opposed to before, irradiation.

script, these anomalous intercepts are probably due to repair of transcriptionterminating lesions; hence, extrapolations of the theoretical dose-response curves should also intercept at the same values as the experimental curves. A comparison of the dose-response curves for the RSV mRNAs and the rRNAs is shown in Fig. 6. The slopes of these curves were used to compute the target sizes of each of the RSV mRNAs, using the inhibition of 23 S rRNA as a standard. This is an acceptable procedure because Giorno and Sauerbier (1976) showed that the rates of formation of transcription terminating lesions for cistrons transcribed by RNA polymerases I and II were nearly the same. The upper part of Fig. 6 shows schematically the relative lengths of the transcriptional units encoding the three viral mRNAs. Within the uncertainties of the data (Fig. 5), the target sizes of all

three species of viral mRNA are the same. The subgenomic mRNAs for env and mc appear to have target sizes somewhat larger than that for mRNABas/@; this might reflect a 10-15s reduction, after uv irradiation, in the rate of processing of the 38 S RNA into the subgenomic mRNA species. Despite the 10-15s variation in target sizes for the three viral RNAs, the experimental data are clearly consistent with the hypothesis that a single promoter exists for all three RSV mRNA species and that the promoter is located at or near the junction of the proviruses and the host cell genome. Processing

of RSV mRNA

Having demonstrated that a single promoter serves to generate all three RSV mRNAs, we explored the pathways that give rise to the two subgenomic mRNAs.

722

HACKETT, VARMUS, AND BISHOP

x)

20

10

10

20

SECONDS

20

UV

FIG. 5. Dose-response curves for RSV mRNA synthesis after irradiation of RSV-infected cells. The effect of uv irradiation on the synthesis of RSV mRNAs was determined as described for Table 1. Panels a, b, and c illustrate the results for mRNAw’ “‘, mRNAa”, and mRNAl respectively. The different symbols represent different experiments; the slopes of the curves and the ordinate intercepts were determined by the method of least squares. The broken lines described theoretical dose-response curves, computed on the assumptions that the RSV transcriptional units were organized as illustrated in Fig. 1B and that the rate of formation of transcriptionterminating lesions on the RSV provirus is the same as on the rRNA cistrons. The Dg7 values, ordinate intercepts, and correlational coefficients for the RSV RNAs are, respectively, for mRNAW’e 13.5 + 1.5 set, 121%, 0.89; mRNAe”“: 12 + 2.5 set, 123% 0.33; mRNA’““: 12.5 + 1.5 set, 123%, 0.92.

Our strategy relied upon the use of the pulse-chase experiments to trace the fate of RNA molecules during the metabolism that follows their synthesis. We grew infected cells in high concentrations of r3H]uridine for 15-30 min, and then “chased” the radioactivity out of the uridine pools by switching to normal media containing glucosamine, unlabeled uridine, and cytidine. Scholtissek (1971), Wertz (19’75), and Herman and Penman (1977) developed this strategy of searching for precursor product relationships among different RNAs. However, when we used the concentrations of glucosamine that they reported (20 mhf in the prepulse media as well as in the chase media), we discovered that the rates of processing of the 41 S and 32 S precursor rRNAs were severely inhibited (unpublished data). Consequently, we examined the feasibility of using lower concentrations of glucosamine to remove the C3H]uridine without interfering with processing. We found that 4 mM glucosamine plus 5 mM unlabeled uridine and cytidine were

iz 2 8

100 80 60 40

5 8

20

\ I

1

mRNAwC

I

20 30 10 SECONDS U.V. FIG. 6. Composite dose-response curves for the RSV mRNAs and rRNAs. Data were taken from Figs. 3 and 5. The upper section of the figure shows the relative target sizes of the transcriptional units based on the slopes of the dose-response curves. The dashed lines indicate the statistical uncertainties of the data. The rRNA cistron was used as a standard, and RSV mRNA target sizes were calculated as described in the text. Shown for comparison is the 33 S (M, 3.3 X 106) RSV RNA subunit isolated from virions.

sufficient to remove effectively [3H]uridine from the uridine pools (Fig. 7) without demonstrable effect on the processing of rRNA (data not shown). In this experiment, we prelabeled the RNA uniformly with [14Cjuridine in order to establish an internal standard for the amount of RNA labeled with 3H during and after the pulse-chase. As shown in Fig. ‘7, the amount of [‘Hluridine incorporated into acid-precipitable RNA during the pulselabeling period was not enhanced by more than 20% as a result of pretreatment of the cells with glucosamine. In all pulsechase experiments presented below we omitted the glucosamine pretreatment step.

GENESIS OF ROW SARCOMA VIRUS MESSENGER RNAs

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60

5

a

!

No Pretreatment

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I

J

40 MIN”TE:o Fig. 7. Pulse-chase experiments in the presence and absence of glucosamine. Infected chick cells were grown for 9 hr in regular medium containing 0.4 mCi/ ml [“CJuridine. To some plates (pretreated, 0) glucosamine was added to a final concentration of 5 mM 0.5 hr before the end of the [‘“Cluridine labeling. The medium in all plates was replaced with medium containing 490 mCi/ml [gH]uridine. At the indicated time points samples were taken for measurement of 3H and “C in acid-precipitable RNA. The ‘H medium was removed from three-fourths of the plates after 40 min and replaced with medium containing either 5mMuridine and 5 m.M cytidine (0) or 5 mM uridine, 5 mMcytidine, 4 tiglucosamine (0, A). To measure the 3H/14C ratios, the media were removed from duplicate plates which were rinsed with isotonic buffer, the cells were lysed with 0.05% (w/v) sodium dodecyl sulfate, and deproteinized by incubation of the lysates with proteinase K (0.2 mg/ml) for 10 min at 37’. Samples (0.1 ml) of the lysate were precipitated in 10% trichloroacetic acid and counted in a liquid scintillation counter. 0, 0, 0, Cultures pretreated with 5 mM glucosamine; A, A, cultures not pretreated.

For analysis of the RSV mRNAs, infected cells were labeled with [32P]orthophosphate for 12 hr; the growth medium was then replaced with fresh medium containing rH]uridine. After 20

723

min, the labeling medium was removed and cells were either harvested or further incubated in media containing glucosamine plus cytidine and uridine. The nucleic acids were isolated from these cultures and the amounts of 3H and 32pin the RSV mRNAs were determined as described for Table 2. The results from one experiment are presented in Table 2 and in graphical form in Fig. 8. These data show that (1) about two-thirds of the intracellular 38 S RSV RNA was lost during a 90-min chase, (2) the rates of accumulation of mRNA”” and mRNA” were the same after the pulse of 3H, (3) very little t3H]mRNAenWand [3H]mRNAWc was made during the first 15 min of incubation, (4) nearly all of the [3H]mRNA”” and [3H]mRNA” was formed during the first 30 min of chasing [in unpublished experiments, 3H-labeled mRNA” and mRNA”” were not detected after 15 min of chasing, suggesting that there is a period of about 30 min-beginning at least 15 min after the synthesis of 38 S RSV RNA-when the subgenomic mRNAs are formed], and (5) the subgenomic RSV mRNAs are stable for at least 60 min after their synthesis. The very large drop in the amount of 3Hlabeled 38 S RSV RNA suggests that more than half of this RNA is either exported from the cell in virions or degraded, since only about 5% of the 3H was recovered in the subgenomic mRNA fractions. DISCUSSION

The Promoter fin- the RSV Provirus Recent work has revealed a possible location for the initiation of RSV RNA synthesis. The provirus of RSV is terminally redundant, with a segment of ca. 330 nucleotides (denoted LTR for “long terminal redundancy”) repeated directly (i.e., in the same orientation) at each end (Hughes et aZ., 1978; Sabran et al., 1979). It has been proposed that the leftward terminal redundancy houses the promotor for viral RNA synthesis (Hughes et al., 19’78; Sabran et al., 1979). Two points of evidence now support this proposal. (i) The composition of the LTRs is such that the 5’ terminus of the viral genome (i.e., the beginning of the coding sequence

HACKETT, VARMUS, AND BISHOP

724

TABLE 2 THE METABOLISM OF RSV RNA”

RNA

mRNA”“/‘“’

mRNA’“”

mRNA””

Chase

3H

=P

(min)

cpm

cpm

‘H/=P

40,220 38,070 31,400 36,560

0.228 0.142 0.085 0.070

4,230 3,520 2,680

0.005 0.060 0.071

0

9160

30 60 90

5420 2660 2570

0 30 60 90

20 210

190 180

3,310

0.054

0

50 200 140 190

4,400 3,010 2,420 2,890

0.011

30 60 90

0.066 0.058 0.066

‘Four plates of cells infected with Pr-B RSV were grown for 12 hr in media containing 1.5 mCi/ml [3’P]orthophosphate. The 32Pmedia was removed, the cells were washed, and fresh media with 800 mCi/ml [3H]uridine was added. After 15 min of incubation, the 3H media was removed from all plates. The cells from one plate were harvested for extraction, and media containing 4 mM glucosamine, 5 m&f uridine, and 5 mM cytidine was added to the other three plates for 30,60, or 90 min incubation at 41”. RSV RNA was analyzed as described for Table 1. Backgrounds in the presence of competitor were: ca. 1000 cpm for [32P]mRNAWu’@; 100 cpm for [“‘PI mRNA’“” and mRNA”‘; 120-230 cpm for [3H]mRNAW’ti; lo-100 cpm for [‘H]mRNA’““; and 30-130 cpm for [3H]mRNA”“.

for viral RNA proper) is located ca. 230 nucleotides in from the left end of the provirus (see Fig. 1). Previous work has raised the possibility that the site of “capping”

I' "

0.16

30

60 90 MINUTES

FIG. 8. Accumulation of RSV mRNAs as a function of time. The ratios of 3H/“P from Table 2 are plotted with respect to the length of time after a 15-min pulse with [‘Hjuridine. A, 38 S RSV RNA; 0, mRNA=“; 0, mRNA”.

of eukaryotic mRNA is identical to the position at which the synthesis of the RNA is initiated (Konkel et al., 1978; Ziff and Evans, 1973). According to this proposal, synthesis of RSV RNA would begin within the leftward LTR of the provirus at the position marking the 5’ terminus of the viral RNA. (ii) A survey of nucleotide sequences located immediately to the left of “capping” sites in eukaryotic genes has identified a series of related nucleotide sequences (or “Hogness Boxes”) that may serve as signals for the initiation of RNA synthesis by RNA polymerase II (Ziff and Evans, 19’78; Gannon et al., 1979). A nucleotide sequence of this sort has been identified in the LTR of RSV, ca. 25 nucleotides to the left of the “capping” site (Czernilofsky et al., 1980; Swanstrom et al., 1980). None of the preceding findings provide a definitive definition of the structural elements required to signal the initiation of mRNA synthesis in eukaryotic cells, i.e., the composition of “promoters” for RNA

GENESIS OF ROUS SARCOMA VIRUS MESSENGER RNA8

polymerase II in eukaryotic cells remains an enigma. For example, recent reports indicate that Hogness Box nucleotide sequences are neither necessary nor sufficient for mRNA synthesis in eukaryotic cells (Baker et al., 1979; Benoist and Chambon, 1980; Grosschedl and Birnstiel, 1980), and candidate Hogness Boxes can be found within genes at points where mRNA synthesis does not initiate (for example, see Van Beveren et al., 1980). It therefore seems erroneous to deduce the location of “promoters” for RNA polymerase II solely on the grounds of local features of nucleotide sequence. The search for such promoters should be supplemented with functional tests, as in the work that we have presented here. Our results sustain previous suggestions that the synthesis of RSV RNA initiates at the junction of the provirus and the host genome (Fig. 1A). In accord with previous results (Quintrell et al., 1980), we have found no evidence that mRNA”” and mRNA” are transcribed from separate, subgenomic proviruses. Our conclusions are based on the finding that the three uv inactivation coefficients for the three RSV mRNAs are nearly the same. The uncertainties in the target size determinations for the viral messengers was lo-20%; we attribute this relatively high value to analytical errors that arise from the low percentage of viral RNA in infected cells. Total viral RNA constituted about 0.2% of the mass of total cellular RNA (Weiss et aZ.,1977; Quintrell et al., 1980), and of this small percentage, ca. 67% was mRNAQ”B’ po’,ca. 9% was mRNA”, and ca. 25% was mRNA” (Quintrell et al., 1980). If the target sizes of the subgenomic RSV mRNAs were smaller than the entire provirus (model B of Fig. 1 and the dashed lines in Fig. 5), then the experimental dose-response curves would have been composed of two components-one resulting from the inactivation of subgenomic transcriptional units (dashed lines in Fig. 5), and the other from degradation products of 38 S RNA synthesis (panel A, Fig. 5). Consequently, the dose-response curves for mRNA”” and mRNA” would have been biphasic and would have been

725

shifted upward toward the broken lines. Our results showing pseudo-first-order dose-response curves for all three viral mRNA species suggest that the data are not appreciably affected by artifact. We therefore conclude that the target sizes for the three RSV mRNAs are approximately the same. Since these RNAs can all arise from a single provirus (Quintrell et al., 1980), a single promoter probably initiates transcription from all of the RSV genes. The statistical error in locating the promoter site for the 38 S RSV RNA was about 10% or 1000 base pairs. Either the RSV provirus is expressed via cellular promoters which lie within 1000 base of the provirus, or RSV encodes its own promoter. Processi~

of RSV RNA

Previous studies with microinjection have shown that subgenomic mRNAs of RSV can be generated by processing of the viral genome (Stacey and Hanafusa, 1978). Our pulse-chase experiments conform to this conclusion by demonstrating that genomic-sized viral RNA is the precursor for both mRNA”” and mRNA” in the infected cell. The subgenomic mRNAs are formed approximately 15-30 min after the appearance of 38 S RNA, a period that corresponds to the processing time of cellular heterogeneous nuclear RNA into cytoplasmic mRNA (Spohr et al., 1974). The available evidence suggests that processing of the retrovirus mRNAs, including the splicing of the 5’ leader sequence onto the protein-coding portions of the mRNAs (Mellon and Duesberg, 19’77; Weiss et at., 1977; Cordell et al., 1978), occurs only in the nucleus (Stacey and Hanafusa, 19’78). Some of the 38 S RNA is converted into mRNA”” and mRNA”; the remainder may be exported from the cell in virions, degraded, or both. Another 25% of the original 38 S transcripts disappears 30-90 min after the formation of 38 S RSV RNA (see Table 2). The data displayed in Fig. 9 suggest that after about 90 min of chasing, the rate of loss of 3H-labeled 38 S RSV RNA was much lower than that during the first 60 min of chase, presumably due to

726

HACKETT, VARMUS, AND BISHOP

stabilization of the 38 S RNA polysomes engaged in the synthesis of Pr76#@’and

veals unusual structure of type C oncornavirus RNA molecules. Cell 7,595-607. BENOIST,C., and CHAMBON,P. (1980). Deletions covPr18F’d ering the putative promoter region of early mRNAs A coroliary to this conclusion is that of simian virus 40 do not abolish T-antigen expresexport of 38 S RNA in virus particles halts sion. Proc. Nat. Acad Sci. USA 77,3365-3369. within 96 min after its synthesis, sugBERGET, S. M., MOORE,C., and SHARP, P. A. (1977). gesting that at some stage the 38 S RNA Spliced segments at the 5’ terminus of adenovirus is segregated into two pools-one for ex2 late mRNA. Proc. Nat. Acad Sci. USA 74,3171port and another for translation as indi3175. cated by a previous study (Levin and Ro- BERK, A. J., and SHARP,P. A. (1977). Sizing and mapsenak, 1976). We cannot say from our data ping of early adenovirus mRNAs by gel electrowhether a third pool, for processing of 38 phoresis of Sl endonuclease digested hybrids. CeU S RNA into subgenomic mRNAs, exists for 12.45-55. a short time in the nucleus. Finally. Table BISHOP, J. M. (1978). Retroviruses. Annu Reu. Biohem. 47.35-33. 2 shows that after 96 min of chase, the ratios of 3H/32P for all three viral RNA BOSSART.W., Nuss, D., L., and PAOLEITI, E. (1978). Effect of UV irradiation on the expression of vacspecies is nearly the same. This identity cinia virus gene products synthesized in a cell-free of ratios suggests that the stabilities of system coupling transcription and translation. J. rnRNAm’&, mRNA”‘, and mRNA”, are Viral 26.673-630. nearly the same; we have not measured CHOU, L. T., GELINAS, BROKER,T., and ROBERTS,R. the half-lives of any of the RSV mRNAs. (1977). An amazing sequence arrangement at the 5’ ends of adenovirus-2 messenger RNA. CeU 12, ACKNOWLEDGMENTS 1-8. COLONNO,R. J., and BANNERJEE,A. K. (1977). MapWe thank Barbara Baker, Janet Lee, and Susan ping and initiation studies on the leader RNA of Weiss for helpful advice and Janet Love and Bertha vesicular stomatitis virus. Viroloss, 77,266~268. Cook for typing the manuscript. This work was supCORDELL, B., WEISS, S. R., VARMUS, H. E., and ported by USPHS Grants CA 12705,CA 1928’7,TrainBISHOP,J. M. (1978). At least 104 nucleotides are ing Grant lT32 CA 09043, and American Cancer Sotransposed from the 5’ terminus of the avian sarciety Grant VC-70. P.B.H. was supported in part by coma virus genome to the 5’ termini of smaller a Damon Runyan-Walter Winchell Fellowship. viral RNAs. CeU 15.75-91. CZERNUFSKY, A. P., DELORBE,W., SWANSTROM,R., REFERENCES VARMUS, H. E., BISHOP, J. M., TISCHER, E., and GOODMAN,H. M. (1980). The nucleotide sequence ATFARDI, G., and AMALDI, F. (1970). Structure and of “c”: An untranslated but conserved domain in synthesis of ribosomal RNA. Annu Rev. Biochmn. the genome of avian sarcoma virus. Nucleic Acid 39,183-226. Res. 8.2967-2934. BAILEY, J. M., and DAVIDSON,N. (1976). Methyl merFAN, H. (1977). RNA metabolism of murine leukemia cury as a reversible denaturing agent for agarose virus: Size analysis of nuclear pulse-labeled virusgel electrophoresis. AnaL Biochem 70.75-35. specific RNA. CeU 11,297~305. BAKER, C. C., HERISSE. J., COURTOIS,G., GAILBERT, FLAMAND, A. and DELAGNEAU, J. F. (1978). TranF., and ZIFF, E. (1979). Messenger RNA for the Ad2 scriptional mapping of rabies virus in tivo. J. Viral DNA binding protein: DNA sequences encoding the 28,518-523. first leader and heterogeneity at the mRNA 5’ end. GANNON, F., O’HARE, K., PERRIN, F., LEPENNEC, Cell 18,569~530. J. P., BENOIST, C., COCHET,M., BREATHNACH,R., BALL, L. A. (1977). Transcriptional mapping of veROYAL, A., GARAPIN, A., CAMI, B. and CHAMBON, sicular stomatitis virus in vivo. J. V&l 21, 411P. (1979). Organization and sequence at the 5’ end 414. of a cloned complete ovalbumin gene. Nature (Lo%BALL, L. A., and WHITE, C. N. (1976). Order of trandon) 278.428-434. scription of genes of vesicular stomatitis virus. GELINAS, R. E., and ROBERTS,R. J. (1977). One promProc. Nat. Acad. Sci. USA 73.442-446. inent 5’-undecanucleotide in adenovirus late mesBEEMON,K., and HUNTER, T. (1977). In vitro transsenger RNAs. CeU 11,533-544. lation yields a possible Rous sarcoma virus src gene GILMORE-HEBERT,M., HERCULES,K.. MCKOMAROMY, product. PTOC.Nat. Acad L&i. USA 74,3302-3306. M., and WALL, R. (1978). Variable and constant BENDER, W., and DAVIDSON, N. (1976). Mapping of regions are separated in the lo-Kbase transcrippoly(A) sequences in the electron microscope re-

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AND BISHOP

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