Vol. 16, No. 6 Printed in U.SA.
JOURNAL OF VIROLOGY, Dec. 1975, p. 1435-1443 Copyright C) 1975 American Society for Microbiology
Characterization of the mRNA of Influenza Virus SYLVIA E. GLASS, DUNCAN McGEOCH,* AND RICHARD D. BARRY Division of Virology, Department ofPathology, University ofCambridge, Addenbrookes Hospital, Cambridge CB2 2QQ, England
Received for publication 7 July 1975
The kinetics of the appearance of influenza mRNA, the distribution of mRNA between free and membrane-associated polyribosomes, its poly(A) content, and the extent to which the genome was transcribed into mRNA early in infection were determined. Polyribosomes were prepared from influenza virus-infected cells labeled for 30-min periods at various times after infection with [3H]uridine. Most of the 3H-labeled RNA extracted from these polyribosomes sedimented as a heterogeneous 8S to 20S peak in sucrose gradients, and it was largely complementary to virion RNA. By the following criteria, the complementary RNA had properties normally ascribed to mRNA: (i) it labeled rapidly with [3H]uridine; (ii) after glutaraldelyde treatment, it banded with polyribosomes in CsCl density gradients; and (iii) it contained poly(A). In chick cells at 37 C, virus mRNA was first detectable at 45 min postinfection and reached its maximal rate of appearance at 2 to 2.5 h postinfection. The free and membrane-bound polyribosomes of infected cells were separated and were found to contain the same classes of mRNA. There was no absolute segregation of mRNA sequences into either polyribosome class although each probably contained distinct ratios of the different mRNA's. From 45 min postinfection onwards, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences complementary to at least 80% of the genome RNA, whereas poly(A)-minus RNA contained sequences complementary to 90 to 100% of the genome. There was no evidence for the temporal control of transcription of influenza mRNA. At 31 C, when virus development was slowed relative to 37 C, complementary RNA first appeared at 1 h postinfection. At this time, total polysomal RNA contained sequences complementary to the whole genome.
Influenza virus particles contain an RNAdependent RNA polymerase, which is thought to transcribe the genome RNA (vRNA) into mRNA in the infected cell (5, 8, 19). Virus complementary RNA (cRNA) was detected in the polyribosome fraction and had some of the properties expected of mRNA (22). However, the possibility that part of the fragmented genome RNA might act directly as mRNA arose with the finding that RNA extracted from virions had some activity in an in vitro translation system; vRNA stimulated the synthesis of a polypeptide resembling viral nucleoprotein (26). The relation of this finding to events in the infected cell remains unclear because it was found subsequently that cRNA, when dissociated from polysomes of canine kidney cells infected with the influenza A strain WSN, was totally complementary to vRNA (12). In this report, we consider the nature of cRNA associated with the polyribosomes of chicken embryo cells infected with the avian influenza A strain, fowl plague.
The influenza virus glycoproteins hemagglutinin and neuraminidase are synthesized exclusively on membrane-bound ribosomes (9, 14, 16). Similarly, it was reported for vesicular stomatitis virus that the 17S class of mRNA, whose translation product is the viral glycoprotein, was associated almost exclusively with membrane-bound polysomes (7, 13). In this study, we investigated the fraction of the influenza genome represented on both the free and the membrane-bound polysomes. It was found that, at the time of maximal cRNA synthesis (2.25 h postinfection), both free and membranebound polysomal poly(A)-containing RNA contained the same cRNA sequences, representing about 80% of the total genome. It has been claimed that selective transcription by the virion-associated transcriptase of three virus genes occurs early in the infection of chick cells by fowl plague virus (27), and data have been produced to support this model (2). We have attempted to measure the extent of virus genome representation in mRNA at very
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early times after infection, and we found that, at 1 h postinfection at 31 C, more than 90% of the genome is represented as cRNA.
J. VIROL.
1% Tween 40 and 0.5% sodium deoxycholate, and then centrifuged for 10 min at 10,000 x g and 4 C. Membrane-bound polysomes were prepared from the supernatant on sucrose gradients. This procedure MATERIALS AND METHODS was used in preference to a detergent treatment of Infection of cells. The influenza A virus, fowl nuclei, which would contaminate the membraneplague, Rostock strain, was used. Virus stocks were bound fraction with free polysomes from any remaingrown in the allantoic cavities of fertile hen eggs. ing whole cells. Work on virus-specific RNA synthesis was carried Method 2 was used in later experiments. The out with confluent monolayer cultures of primary combined supernatants after nuclear washing were chicken embryo cells. Cultures were infected at a fractionated in a discontinuous sucrose-D20 gramultiplicity of 5 PFU/cell for 30 min at room temper- dient system (B. Mechler and P. Vassalli, J. Cell ature, washed with phosphate-buffered saline, and Biol., in press). The membrane-associated polyincubated with prewarmed minimal essential me- somes floated away from the denser free polysomes. dium containing 2% calf serum. The zero time of RNA was prepared from the appropriate fractions as infection was taken as the time of removal of the described above. This method has been shown to inoculum. RNA was radiolabeled in a medium con- give very low levels of cross-contamination of the taining [3H]uridine or [3H]adenosine at 50 j,Ci/ml. two polysome classes. The two methods gave very Preparation of total polyribosomal RNA. Our similar recoveries and proportions of [3H]uridinemethod for preparation of total polyribosomal RNA labeled RNA in the two classes of polysomes. was based on that of Hulse and Wettstein (15). The Density gradient analysis of polyribosomes. medium was removed, and the cells were washed RNA was labeled by incubation of cells for 24 h with twice with semifrozen phosphate-buffered saline, 0.1 ,uCi of [14C]uridine per ml. Cultures were inscraped from the dish, and centrifuged at 800 x g for fected and pulse labeled with [3Hluridine, and polyri5 min at 4 C. The cells were disrupted in a Dounce bosomes were prepared on sucrose gradients as homogenizer after suspension in reticulocyte stand- above, except that 10 mM sodium cacodylate, pH ard buffer (10 mM Tris-hydrochloride [pH 7.4], 10 7.4, was substituted for Tris-hydrochloride. PolymM KCl, 1.5 mM MgCl2) plus 0.01% diethylpyrocar- some fractions >808 were pooled and fixed with bonate and swelling for 10 min at 0 C. The nuclear glutaraldehyde as described by Baltimore and fraction was removed by centrifuging at 800 x g for Huang (3) and then centrifuged to equilibrium (100,45 s at 4 C. The nuclei were washed twice with 000 x g for 16 h at 4 C) in 30 to 55% (wt/wt) CsCl reticulocyte standard buffer plus 0.01% diethylpyro- gradients containing 10 mM sodium cacodylate (pH carbonate, and all three supernatants were pooled 7.4), 10 mM NaCl, 1.5 mM MgCl2, and 0.8% Brij-58. and centrifuged at 800 x g and 4 C for 1.5 min. The RNA was prepared from gradient fractions by desaltsupernatant was made 1% in Tween 40 and 0.5% in ing on an agarose column (Bio Gel AO.5M) equilisodium deoxycholate, centrifuged for 10 min at 10,- brated with 10 mM Tris-hydrochloride, pH 7.4, and 1 000 x g and 4 C, layered on a 15 to 55% (wt/vol) mM EDTA and then by Pronase treatment as above. sucrose gradient in a solution of 50 mM Tris-hydroPreparation of poly(A)-containing RNA. RNA chloride (pH 7.5), 25 mM KCl, 5 mM MgCl2, and preparations were dissolved in a solution of 10 mM 0.01% diethylpyrocarbonate, and centrifuged for 15 Tris-hydrochloride (pH 7.6), 0.4 M NaCl, 1 mM h at 30,000 x g and 4 C. The gradient was fraction- EDTA, 10% (vollvol) 0.1% sodium dodecyl sulfate ated and the profile of the absorbancy at 254 nm and passed through a 0.2-ml oligo(dT)-cellulose col(A254) was recorded. The polysomes in the region umn (11). After the eluted RNA was washed with greater than 80S were pooled as the source of the above buffer, it was pooled as poly(A)-minus. mRNA. Care was taken not to include 80S mono- Poly(A)-containing RNA was eluted with a solution somes since influenza virus ribonucleoprotein sedi- of 10 mM Tris-hydrochloride (pH 7.6), 1 mM EDTA, ments around 70S (23). The pooled fractions were and 0.1% sodium dodecyl sulfate. made 0.5% in sodium dodecyl sulfate and precipiRNA-RNA annealing procedures. RNA samples tated with 2 volumes of EtOH. The precipitate was were dissolved in 2x SSC (lx SSC is 0.15 M NaCl dissolved in a solution of 0.1 M Tris-hydrochloride plus 15 mM sodium citrate, pH 7.0). Annealing of (pH 7.5), 50 mM NaCl, 10 mM EDTA, 4 lAg of polyvi- desired mixtures was carried out in 50- or 75-!AI volnyl sulfate per ml, and 0.5% sodium dodecyl sulfate umes in sealed glass ampoules for 18 h at 68 C. and digested with 0.5 mg of Pronase per ml for 30 Samples were recovered in 1.0 ml of 2x SSC. A 0.5min at 37 C (1, 18). After extraction with chloro- ml portion was incubated with 0.5 ml of RNase A form-isoamyl alcohol (24:1, vol/vol), the RNA was (100 ,ug/ml) and RNase T1 (100 U/ml) in 2x SSC for precipitated with 2.5 volumes of EtOH. 30 min at 37 C. The RNase-resistant fraction was Preparation of free and membrane-bound poly- then determined by precipitating the sample and an ribosomes.Two methods were used for the prepara- equivalent undigested sample with 2 ml of 1 N pertion of free and membrane-bound polyribosomes. chloric acid, with 100 jAg of heat-denatured calf thyMethod 1 is a modification of that described above. mus DNA as carrier. The precipitate was collected After the nuclear fraction was washed, the com- on a glass fiber filter, washed with 0.5 N trichloroabined supernatants were centrifuged for 10 min at cetic acid and then EtOH, and dried, and the radioac10,000 x g and 4 C. Free polysomes were prepared tivity was measured in a scintillation spectrometer. from the supernatant on sucrose gradients as above. Preparation of radiolabeled virion RNA. HighThe pellet was dissolved in reticulocyte standard specific-activity 32P-labeled vRNA was prepared as buffer plus 0.1% diethylpyrocarbonate, treated with follows. Chicken embryo cell monolayers in phos-
INFLUENZA VIRUS mRNA
VOL. 16, 1975 phate-free medium 199 were infected with about 0.5 PFU/cell and incubated for 27 h at 37 C in the presence of 200 E&Ci of [32P]P, per ml. The medium was collected, chilled, and centrifuged for 10 min at 10,000 x g and 4 C. The supernatant was centrifuged for 30 min at 120,000 x g and 4 C, and the pellet was suspended in a 0.5-ml solution of 0.1 M NaCl, 10 mM Tris-hydrochloride (pH 7.4), and 1 mM EDTA (NTE), layered on a 15 to 60% (wt/vol) sucrose gradient in NTE (10 ml), and centrifuged for 30 min at 150,000 x g and 4 C. The visible virus band was collected, diluted with 1 volume of water, layered on a 10-ml gradient of potassium tartrate (20 to 45%, wt/vol) in 10 mM Tris-hydrochloride (pH 7.4), and centrifuged for 12 h at 100,000 x g and 4 C. The virus band was diluted with NTE and centrifuged for 30 min at 150,000 x g and 4 C. RNA was extracted by suspending the virus pellet in 0.5 ml of NTE containing 0.5% sodium dodecyl sulfate and 80 itg of tRNA, extracting it twice with NTE-saturated phenol, and then precipitating it with EtOH. 32P-labeled vRNA preparations had specific activities around 1.4 x 106 counts/min per jtg. 3H-labeled vRNA was prepared by the growth of virus in purine- and pyrimidine-free medium 199 supplemented with 1 mCi of [3H]uridine per ml and 1 mCi of [3H]adenosine per ml. 3H-labeled vRNA had a specific activity of 5 x 106 counts/min per Slg.
RESULTS Detection of cRNA in polysomes. All results refer to virus growth at 37 C, except for one set at 31 C described later. Fowl plague virus-infected chick cells were labeled for 30min periods at various times after infection
1437
with [3H]uridine, and polyribosomes were prepared (Fig. 1). RNA was extracted from polysomes and tested for self-annealing in the presence of excess unlabeled vRNA. From 45 min onwards, [3H]RNA complementary to vRNA was detectable (Fig. 2). The rate of appearance of cRNA reached a maximum at 2 to 2.5 h postinfection. At early stages of infection, there was little self-annealing of [3HIRNA, indicating a low content of vRNA in the polysome preparations. However, as infection progressed, increasing amounts of vRNA were present by this criterion. At all stages of infection, high levels of self-annealing (25 to 40%) were observed with [3H]RNA prepared from fractions 80S was analyzed in this study of mRNA. Typically, about four times more 3H label was incorporated into free polysomal RNA than into membrane-bound polysomal RNA. To determine what proportion of 3H-labeled polysomal RNA was cRNA and also to estimate its size, RNA was extracted from free and membrane-bound polysomes prepared from 3Hpulse-labeled chick cells at 2.25 h postinfection and analyzed on sucrose rate zonal density gradients. The 3H-labeled RNA sedimented heterogeneously (range 3S to 20S) with a peak at 13S (Fig. 3). RNA was recovered from each fraction of each gradient by ethanol precipitation and 08
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FIG. 1. Fractionation of polyribosomes. Free polyribosomes were prepared from cells 2.25 h postinfection, sedimented through a sucrose gradient, and colkected into fractions. Stable RNA was labeled with [14C]uridine (16 h at 01 ACilmi) and rapidly labeled RNA with [3H]uridine (30 min at.50 pCi/ml). Symbols: , An, *, 3H counts per minute; 0, 14C counts per minute.
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GLASS, McGEOCH, AND BARRY
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level of annealing to vRNA, whereas the [3H]RNA present in less dense structure shows a high level of self-annealing, with little or no additional annealing to added vRNA. This demonstrates that most of the [3HIcRNA is closely associated with ribosomes, in contrast to vRNA that is present in less dense structures, presumIl A I-
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FIG. 2. Synthesis of polyribosomal virus complementary RNA. Cells were labeled with [3H]uridine for 30 min at various times after infection. Membrane-bound and free polyribosomes were prepared. Self-annealing and cRNA content of the [3HIRNA were measured. Each point represents the end time of a 30-min labeling period. Symbols: 0, percentage of self-annealing of [3H]RNA; *, percentage of 3H in cRNA as determined by annealing to an excess of vRNA.
annealed with an excess of virion RNA. Most of the rapidly labeled RNA was cRNA (Fig. 3) and of a size comparable to the constituents of the fragmented vRNA. We treated isolated polysomes with puromycin in attempts to dissociate and purify mRNA. The [3H]RNA was released and sedimented in sucrose rate gradients at 40S to 60S. A possible explanation for this result is that influenza mRNA in chick cells occurs as a nucleoprotein. Polyribosome-associated messenger ribonucleoproteins have been reported (20). Banding of polysomes and cRNA in CsCl gradients. Cosedimentation ofcRNA with polyribosomes in sucrose gradients does not show conclusively that the cRNA is mRNA. To demonstrate that the [3H]cRNA was closely associated with ribosomes, we fixed polysome preparations with glutaraldehyde and centrifuged them to equilibrium in CsCl density gradients (3) (Fig. 4). In all cases most of the rapidly labeled RNA banded with rRNA (which had been prelabeled with [l4Cluridine) at a density of 1.5 g/ml, as expected for polysomes (3). Some [3HIRNA was present in less dense structures, presumably ribonucleoprotein particles (17), especially in free polysome preparations and at later times. With polysomes prepared 4.5 h after infection and fractionated in CsCl, RNA was extracted from pooled fractions and analyzed for self-annealing and annealing to vRNA (Table 1). For both free and membrane-bound
:i
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polysomes, the genuine polysomal [3HIRNA shows a low level of self-annealing and a high
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FIG. 3. Sedimentation ofpolyribosomal virus complementary RNA in sucrose rate gradients. Free and membrane-bound polyribosomes were prepared from cells 225 h postinfection after 30-min labeling with [3H]uridine 450 ACi/ml). Polysomal RNA was extracted and sedimented on 17 ml of 5 to 20% (wtlvol) sucrose density gradients in 10 mM Tris-hydrochloride (pH 7.4)-0.1 M NaCl-1 mM EDTA-02% sodium dodecyl sulfate buffer at 57,000 x g for 14 h at 22 C. Gradients were collected in 1-ml fractions from the top, and the optical density profile was recorded at 254 nm. 100-pli portions were taken from each fraction to determine trichloroacetic acid-insoluble radioactivity. Each fraction was precipitated with 2 volumes EtOH, collected, and hybridized to vRNA (25 jg/ml). Symbols: A2S4 0, 3H counts per -,
minute hybridized to vRNA;
0,
total 3H counts per
minute. (A) 225 h postinfection, free polysomal RNA; (B) 225 h postinfection, membrane-bound RNA.
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FIG. 4. Fractionation of polyribosomes in CsCl gradients. Cells were labeled as for Fig. 1. Membranebound and free polyribosomes were prepared at 2.25 and 4.25 h postinfection and fractionated by sedimentation through a sucrose gradient. Fractions ofgreater than 8JS were fixed with glutaraldehyde and centrifuged to equilibrium in CsCL gradients. Symbols: 0, 3H counts per minute; 0, 14C counts per minute; A, density grams per cubic centimeter. (A) 2.25 h postinfection; free polysomes, upper panel; membrane-bound polysomes, lower panel. (B) 4.25 h postinfection; free polysomes, upper panel; membrane-bound polysomes, lower panel. TABLE 1. Density gradient fractionation of rapidly labeled RNA associated with polysomesa Membrane-bound polysomes
Free polysomes Fraction
Polysomes, density 1.5 g/ml Ribonucleoprotein, density 1.34 to 1.39 g/ml
% of total 3H
71.7 28.3
SelfAnnealing annealing to vRNA 5.4 24.3
82.2 19.2
% Of % of
69.2 31.8
SelfAnnealing annealing to vRNA
4.0 18.4
69.9 23.7
a Polyribosomes labeled with [3H]uridine were prepared 4.5 h postinfection on sucrose gradients. Fractions >80s were fixed with glutaraldehyde and centrifuged to equilibrium in CsCl. RNA was extracted from appropriate fractions and tested for self-annealing and annealing to excess vRNA.
ably ribonucleoprotein particles (17). Poly(A) sequences in cRNA. RNA prepared from polysomes 2.25 h postinfection and labeled with [3H]uridine or [3H]adenosine was fractionated on oligo(dT)-cellulose columns (11), and the RNase resistance of the fractions was measured (Table 2). [3H]uridine-labeled cRNA from both membrane-bound and free polysomes gave around 50% binding to oligo(dT)-cellulose, with low levels (2 to 4%) of intrinsic RNase resist-
ance. With [3H]adenosine-labeled RNA, the material binding to oligo(dT)-cellulose had an intrinsic RNase resistance of 22 to 28%. We conclude that about half of the cRNA contained covalently linked poly(A) sequences. Proportion of the virus genome represented in cRNA. The fraction of the influenza virus genome RNA represented in preparations of polysomal RNA was estimated as follows. A series of mixtures was set up in which a con-
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GLASS. McGEOCH, AND BARRY
TABLE 2. Poly(A) content of polyribosomal RNAa % Of Polysomes
% cRNA of 3H
in
cRNA
% 3H RNase rssac
poly(A)
Free [3H]A label [3H]U label
66.7 64.4
54.1 53.4
22.0 2.7
2.3 2.9
Membranebound [3H]A label [3H]U label
70.5 62.3
49.6 41.8
28.0 4.1
2.5 3.7
J. VIROL.
iments with membrane-bound and free polysomal poly(A)-containing RNA from 4.5 h postinfection gave similar results. cRNA present early in infection. RNA was prepared from total polysomes 0.75 h postinfection, which is the earliest time at which cRNA had previously been detected (4). To analyze the expected low levels of cRNA we used 3H-labeled vRNA of specific activity 5 x 106 counts/min per ,ug. A poly(A)-plus fraction of cRNA annealed 92% of the input vRNA. Total polysomal RNA annealed 82%, without reaching a clear plateau at the highest concentration available (data not
shown). After previous work by others (2, 27), we Cells were labeled with [3H]adenosine ([3HIA) or examined, by this method, early events of infec[3Hluridine ([H]U) for 30 min and at 2.25 h postinfection polyribosomes were prepared (method 1) and tion at 31 C. Total polysomal RNA was preRNA was extracted. cRNA content was assayed by pared from cells maintained at 31 C for 1 and 2 annealing samples of [3H]RNA to an excess of h after infection. At both times >90% of the vRNA. Samples were fractionated on oligo(dT)-cellu- genome is represented as cRNA (Fig. 6). lose, and cRNA content and RNase resistance were determined. DISCUSSION Virus mRNA was first detected at 45 min stant amount of 32P-labeled vRNA, usually postinfection, a time when synthesis of total 1,000 counts/min, was hybridized to a large cRNA is just starting (4), so evidently nascent range of concentrations of an unlabeled polysomal RNA preparation. As increasing amounts of cRNA are annealed to a constant amount of vRNA, the final percentage of vRNA found in an RNase-resistant form should represent the proportion of the genome with complementary sequences in polysomal RNA. We first analyzed, by this method, polysomal RNA prepared from cells 2.25 h after infection. RNA from both free and membrane-bound polysomes contained sequences complementary to 0 90 to 100% of the virus genome RNA (Fig. 5A). iO POLYRIBOSOMAL RNA CONCENTRATION mg/ml When free polysomal RNA was fractionated into poly(A)-containing and poly(A)-minus LA 100 B RNA, the poly(A)-minus RNA annealed 100% 80 and the poly(A)-plus RNA annealed 80% of the 4-0-. vRNA (data not shown). These preparations of polysomal RNA were 5Q 60 6 made by method 1 above. We wished to exclude 40 the possibility that the results were affected by cross-contamination of the two polysome 20 classes. Preparations of polysomes were made by method 2, which has been shown to give very 30 40 100 I50 little cross-contamination (Mechler and VasPOLYRIBOSOMAL alli, in press). In this case, only RNA containFIG. 5. Representation of genome sequences in ing poly(A) was used since preparations of [32P]vRNA (1,000 counts/min) was annealed poly(A)-minus RNA made by this method con- cRNA. a range of concentrations of unlabeled polyribosotain vRNA, which could lead to artifactual re- to mal RNA from2 25 h postinfection, and RNase resistsults. As shown in Fig. 5B, both classes anneal ance of the vRNA was determined. (A) Total polyriboabout 80% of the [32P]vRNA. Addition experi- somal RNAs. Symbols: *, total free polysomal RNA; ments in which mixtures of the two polysomal 0, total membrane-bound polysomal RNA. (B) RNAs were annealed to [32P]vRNA gave little Poly(A)-containing RNAs. Symbols: 0, from free or no additivity; 80 to 85% was annealed. Exper- polysomes; 0, from membrane-bound polysomes. a
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RNA CONCENTRATION
pg/Inl
250
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INA CONCENTRATI ON mg/I
FIG. 6. Representation ofgenome sequences in polysomal RNA during infection at 31 C. [3H]vRNA was used, as for Fig. 4. (A) Total polyribosomal RNA from 1 h postinfection at 31 C. (B) Total polyribosomal RNA from 2 h postinfection at 31 C.
cRNA must be rapidly processed into polyribosomal RNA. The maximal rate of appearance of virus mRNA, as measured by 30-min pulses with [3H]uridine, was at 2 to 2.5 h postinfection. These data compare reasonably with previous measurements (24) on the appearance of total cRNA in fowl plague virus-infected cells. At 2 to 2.5 h postinfection, 60 to 70% of the rapidly labeled polysomal RNA is virus cRNA; evidently a large proportion of the synthetic capacity of the cell is turned over to the synthesis of virus-specific products. Both complementary and virus-strand RNA were detectable in polysome fractions from sucrose gradients, the latter increasingly at later times. However, only cRNA behaved as messenger when fixed polysome preparations were fractionated by buoyant density in CsCl. Around 50% of the polysomal cRNA contained poly(A) sequences by the criterion of binding to oligo(dT)-cellulose. There was no evidence of vRNA in the poly(A)-containing fractions. We conclude, in agreement with previous results (12, 22), that virus cRNA behaves as mRNA by the following criteria: (i) rapid labeling with [3H]uridine in polyribosomal fractions of sucrose gradients, (ii) banding with polyribosomes in CsCl density gradients, and (iii) pos-
session of a poly(A) sequence in the molecule. The vRNA found with polysomes in sucrose gradients is probably progeny vRNA, as ribonucleoprotein particles, adventitiously associated with ribosomes (17, 20, 21). We examined the fraction of the genome represented in various polysomal cRNA preparations. Since the influenza virus genome is segmented into RNA pieces, each coding for one protein (10, 28), we are analyzing a population of up to about eight monocistronic mRNA's. At 2.25 h postinfection, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences representing about 80% of the total genome. Mixed annealing experiments showed little or no increase (80 to 85%). Therefore, both polysome classes contain the same 80% of the genome. This result was unexpected. It is considered that the virus glycoproteins hemagglutinin and neuraminidase are synthesized exclusively on membrane-bound ribosomes (9, 14, 16). The genes for these proteins comprise about 30% of the virus genome (10, 28). We, therefore, expected the membranebound polysomal RNA to contain a fraction, 30% of the total, not found in free polysomal RNA. (Such a situation is found with the rhabdovirus vesicular stomatitis virus [7, 131.) The polysome preparations used were made by a method giving minimum cross-contamination of the two classes (Mechler and Vassalli, in press). Similar results were obtained from RNA extracted 4.5 h postinfection. We conclude that there is no absolute segregation of mRNA sequences into the two polysome classes, but our analysis does not discriminate the relative proportions of various mRNA species in a preparation, so it is possible that each polysome class contains distinct ratios of the different mRNA's. This is suggested by Fig. 4A: although low concentrations offree and membrane-bound polysomal RNA give an identical annealing response, much more membrane-bound than free RNA is required to anneal to completion. Shafritz (25) has demonstrated a similar situation in rabbit liver polysomes, where free and membrane-bound fractions each contain mRNA's not normally translated in that class. We were interested in examining the mRNA species detectable at very early times after infection. Analysis of polysomal RNA from 45 min postinfection showed that total polysomal RNA contained sequences complementary to 82% of the virus genome, whereas poly(A)-containing RNA could anneal 92% of the input vRNA. Since, at this time, cRNA synthesis has just started (4), these experiments indicate that, from a very early stage of infection on-
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wards, at 37 C almost all of the virus genome is ity of the various mRNA species, and some transcribed. Skehel (27) has analyzed the syn- sequences could be transcribed much less than thesis of influenza virus-specified polypeptides others early in infection. at early times after infection. He found that at In polysomal RNA obtained at 2.25 h after 31 C, with virus development slowed relative to infection (Fig. 5), we found that poly(A)-contain37 C, some proteins appeared earlier than oth- ing RNA contained sequences complementary ers, suggesting the possibility of selective to around 80% of the genome RNA, whereas mRNA production. Avery and Dimmock (2) in- poly(A)-minus RNA contained sequences comvestigated this possibility by annealing plementary to 90 to 100% of the genome. This [32P]vRNA to total RNA from cells infected result was not due to differences in input cRNA with influenza virus for 2 h at 31 C. They could concentrations. We have no satisfactory expladetect cRNA sequences complementary to only nation for the phenomenon. Conceivably, some 25% of the genome, as opposed to 50% later in poly(A)-minus RNA might not be mRNA but infection, and concluded that a temporal con- might be part of an RNA replication mechatrol of transcription does indeed operate at nism. By the criterion of poly(A) association, at 31 C. In contrast, we found that total polysomal least 80% of the genome codes for mRNA. RNA preparations from 1 and 2 h after infection ACKNOWLEDGMENTS at 31 C contained sequences complementary to thank H. Patmore, R. Farrell, and N. Kitron for We more than 90% of the genome RNA (Fig. 6). technical assistance, Our experimental technique differs from that of of the manuscript. and B. W. J. Mahy for a critical review Avery and Dimmock in two important respects. S. E. G. is a Medical Research Council predoctoral stuFirst, we used radiolabeled vRNA of about 60- dent. This work was supported by grants from the Medical fold higher specific activity, so our experiments Research Council. are more sensitive. Second, we analyzed polyLITERATURE CITED somal RNA containing little or no vRNA, as opposed to total cell RNA, which must contain all 1. Aloni, Y., and G. Attardi. 1971. Symmetrical in vivo transcription of mitochondrial DNA in HeLa cells. of the infecting vRNA. With total cell RNA Proc. Natl. Acad. Sci. U.S.A. 68:1757-1761. preparations from early stages of infection, the 2. Avery, R. J., and N. J. Dimmock. 1975. Temporal conpresence of unlabeled vRNA could lead to artitrol of transcription of influenza virus RNA. Virology 64:409-414. factually low levels of annealing to labeled 3. Baltimore, D., and A. S. Huang. 1968. Isopycnic separavRNA. tion of subcellular components from poliovirus-inOur attempts to detect transcriptional confected and normal HeLa cells. Science 163:572-574. trol were carried out at 31 C (Fig. 6) and were 4. Bean, W. J., and R. W. Simpson. 1973. Primary trandesigned to measure the amount of representascription of the influenza virus genome in permissive cells. Virology 56:646-651. tion of the virus genome in mRNA, rather than D. H. L., J. F. Obijeski, and R. W. Simpson. to measure the quantity of mRNA present. The 5. Bishop, 1971. Transcription of the influenza ribonucleic acid amount of polyribosomal RNA obtained 1 h genome by a virion polymerase. I. Optimal conditions postinfection needed to saturate 3H-labeled for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase. J. Virol. 8:66-73. vRNA (Fig. 6A) suggests that even the most G. 1971. Release, identification and isolation of frequently represented sequences of virus 6. Blobel, messenger RNA from mammalian ribosomes. Proc. mRNA were present in only small amounts at 1 Natl. Acad. Sci. U.S.A. 68:832-835. h after infection at 31 C. We believe, therefore, 7. Both, G. W., S. A. Moyer, and A. K. Banerjee. 1975. Translation and identification of the viral mRNA that at this stage of infection we are looking at species isolated from subcellular fractions of vesicuan intrinsically incomplete process and do not lar stomatitis virus-infected cells. J. Virol. 15:1012wish to place much weight on these results. 1019. However, at 1 h after infection at 31 C, the 8. Chow, N., and R. W. Simpson. 1971. RNA-dependent RNA polymerase activity associated with virions and hybridization curve was biphasic and required subviral particles of myxoviruses. Proc. Natl. Acad. a large amount (12 mg/ml) of polyribosomal Sci. U.S.A. 68:752-756. RNA to saturate the 3H-labeled vRNA (Fig. 9. Compans, R. W. 1973. Influenza virus proteins. II. Asso6A). These data suggest that about half of the ciations with components of the cytoplasm. Virology genome is represented in much greater quan- 10. 51:56-70.R. Compans, W., and P. W. Choppin. 1975. Reproductity than the other half. Nevertheless, this tion of myxoviruses, p. 179-252. In H. Fraenkel-Conof sequence representation gross asymmetry rat and R. R. Wagner (ed.), Comprehensive virology, vol. 4. Plenum Press, New York. had disappeared by 2 h postinfection at 31 C. We conclude that influenza virus does not ex- 11. Edmonds, M., and M. G. Caramela. 1969. The isolation and characterization of adenosine-monophosphatehibit a temporal control of transcription, where rich polynucleotides synthesized by Ehrlich ascites "temporal control" means that some sequences cells. J. Biol. Chem. 244:1314-1324. are not transcribed until later times. However, 12. Etkind, P. R., and R. M. Krug. 1974. Influenza viral messenger RNA. Virology 62:38-45. our technique does not demonstrate equimolar-
VOL. 16, 1975 13. Grubman, M. J., E. Ehrenfeld, and D. F. Summers. 1974. In vitro synthesis of proteins by membranebound polyribosomes from vesicular stomatitis virusinfected HeLa cells. J. Virol. 14:560-571. 14. Hay, A. J. 1974. Studies on the formation of the influenza virus envelope. Virology 60:398-418. 15. Hulse, J. L., and F. 0. Wettstein. 1972. Two separable cytoplasmic pools of native ribosomal subunits in chick embryo tissue culture cells. Biochim. Biophys. Acta 269:265-275. 16. Klenk, H. D., W. W6llert, R. Rott, and C. Scholtissek. 1974. Association of influenza virus proteins with cytoplasmic fractions. Virology 57:28-41. 17. Krug, R. M. 1972. Cytoplasmic and nucleoplasmic viral RNPs in influenza virus-infected MDCK cells. Virology 50: 103-113. 18. Miller, L., and G. Knowland. 1970. Reduction of ribosomal RNA synthesis and ribosomal RNA genes in a mutant of Xenopus laevis which organizes only a partial nucleolus. II. The number of ribosomal RNA genes in animals of different nucleolar types. J. Mol. Biol. 53:329-338. 19. Penhoet, E., H. Miller, M. Doyle, and S. Blatti. 1971. RNA-dependent RNA polymerase activity in influenza virions. Proc. Natl. Acad. Sci. U.S.A. 68:13691371. 20. Penman, S., C. Vesco, and M. Penman. 1968. Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and po-
INFLUENZA VIRUS mRNA
21.
22.
23.
24.
25.
26.
27.
28.
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lyribosome-associated messenger RNA in HeLa cells. J. Mol. Biol. 34:49-69. Perry, R. P., and D. E. Kelley. 1968. Messenger RNAprotein complexes and newly synthesized ribosomal subunits: analysis of free particles and components of polyribosomes. J. Mol. Biol. 35:37-59. Pons, M. W. 1971. Isolation of influenza virus ribonucleoprotein from infected cells. Demonstration of the presence of negative-stranded RNA in viral RNP. Virology 46:149-160. Pons, M. W. Studies on the replication of influenza virus RNA. Virology 47:823-832. Scholtissek, C., and R. Rott. 1970. Synthesis in vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40:989-996. Shafritz, D. A. 1974. Evidence for nontranslated messenger ribonucleic acid in membrane-bound and free polysomes of rabbit liver. J. Biol. Chem. 249:89-93. Siegert, W., G. Bauer, and P. H. Hofschneider. 1973. Direct evidence for message activity of influenza virion RNA. Proc. Natl. Acad. Sci. U.S.A. 70:29602963. Skehel, J. J. 1973. Early polypeptide synthesis in influenza virus-infected cells. Virology 56:394-399. White, D. 0. 1974. Influenza viral proteins: identification and synthesis. Curr. Top. Microbiol. Immunol. 63:1-48.
JOURNAL OF VIROLOGY, Dec. 1975, p. 1435-1443 Copyright C) 1975 American Society for Microbiology
Characterization of the mRNA of Influenza Virus SYLVIA E. GLASS, DUNCAN McGEOCH,* AND RICHARD D. BARRY Division of Virology, Department ofPathology, University ofCambridge, Addenbrookes Hospital, Cambridge CB2 2QQ, England
Received for publication 7 July 1975
The kinetics of the appearance of influenza mRNA, the distribution of mRNA between free and membrane-associated polyribosomes, its poly(A) content, and the extent to which the genome was transcribed into mRNA early in infection were determined. Polyribosomes were prepared from influenza virus-infected cells labeled for 30-min periods at various times after infection with [3H]uridine. Most of the 3H-labeled RNA extracted from these polyribosomes sedimented as a heterogeneous 8S to 20S peak in sucrose gradients, and it was largely complementary to virion RNA. By the following criteria, the complementary RNA had properties normally ascribed to mRNA: (i) it labeled rapidly with [3H]uridine; (ii) after glutaraldelyde treatment, it banded with polyribosomes in CsCl density gradients; and (iii) it contained poly(A). In chick cells at 37 C, virus mRNA was first detectable at 45 min postinfection and reached its maximal rate of appearance at 2 to 2.5 h postinfection. The free and membrane-bound polyribosomes of infected cells were separated and were found to contain the same classes of mRNA. There was no absolute segregation of mRNA sequences into either polyribosome class although each probably contained distinct ratios of the different mRNA's. From 45 min postinfection onwards, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences complementary to at least 80% of the genome RNA, whereas poly(A)-minus RNA contained sequences complementary to 90 to 100% of the genome. There was no evidence for the temporal control of transcription of influenza mRNA. At 31 C, when virus development was slowed relative to 37 C, complementary RNA first appeared at 1 h postinfection. At this time, total polysomal RNA contained sequences complementary to the whole genome.
Influenza virus particles contain an RNAdependent RNA polymerase, which is thought to transcribe the genome RNA (vRNA) into mRNA in the infected cell (5, 8, 19). Virus complementary RNA (cRNA) was detected in the polyribosome fraction and had some of the properties expected of mRNA (22). However, the possibility that part of the fragmented genome RNA might act directly as mRNA arose with the finding that RNA extracted from virions had some activity in an in vitro translation system; vRNA stimulated the synthesis of a polypeptide resembling viral nucleoprotein (26). The relation of this finding to events in the infected cell remains unclear because it was found subsequently that cRNA, when dissociated from polysomes of canine kidney cells infected with the influenza A strain WSN, was totally complementary to vRNA (12). In this report, we consider the nature of cRNA associated with the polyribosomes of chicken embryo cells infected with the avian influenza A strain, fowl plague.
The influenza virus glycoproteins hemagglutinin and neuraminidase are synthesized exclusively on membrane-bound ribosomes (9, 14, 16). Similarly, it was reported for vesicular stomatitis virus that the 17S class of mRNA, whose translation product is the viral glycoprotein, was associated almost exclusively with membrane-bound polysomes (7, 13). In this study, we investigated the fraction of the influenza genome represented on both the free and the membrane-bound polysomes. It was found that, at the time of maximal cRNA synthesis (2.25 h postinfection), both free and membranebound polysomal poly(A)-containing RNA contained the same cRNA sequences, representing about 80% of the total genome. It has been claimed that selective transcription by the virion-associated transcriptase of three virus genes occurs early in the infection of chick cells by fowl plague virus (27), and data have been produced to support this model (2). We have attempted to measure the extent of virus genome representation in mRNA at very
1435
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GLASS, McGEOCH, AND BARRY
early times after infection, and we found that, at 1 h postinfection at 31 C, more than 90% of the genome is represented as cRNA.
J. VIROL.
1% Tween 40 and 0.5% sodium deoxycholate, and then centrifuged for 10 min at 10,000 x g and 4 C. Membrane-bound polysomes were prepared from the supernatant on sucrose gradients. This procedure MATERIALS AND METHODS was used in preference to a detergent treatment of Infection of cells. The influenza A virus, fowl nuclei, which would contaminate the membraneplague, Rostock strain, was used. Virus stocks were bound fraction with free polysomes from any remaingrown in the allantoic cavities of fertile hen eggs. ing whole cells. Work on virus-specific RNA synthesis was carried Method 2 was used in later experiments. The out with confluent monolayer cultures of primary combined supernatants after nuclear washing were chicken embryo cells. Cultures were infected at a fractionated in a discontinuous sucrose-D20 gramultiplicity of 5 PFU/cell for 30 min at room temper- dient system (B. Mechler and P. Vassalli, J. Cell ature, washed with phosphate-buffered saline, and Biol., in press). The membrane-associated polyincubated with prewarmed minimal essential me- somes floated away from the denser free polysomes. dium containing 2% calf serum. The zero time of RNA was prepared from the appropriate fractions as infection was taken as the time of removal of the described above. This method has been shown to inoculum. RNA was radiolabeled in a medium con- give very low levels of cross-contamination of the taining [3H]uridine or [3H]adenosine at 50 j,Ci/ml. two polysome classes. The two methods gave very Preparation of total polyribosomal RNA. Our similar recoveries and proportions of [3H]uridinemethod for preparation of total polyribosomal RNA labeled RNA in the two classes of polysomes. was based on that of Hulse and Wettstein (15). The Density gradient analysis of polyribosomes. medium was removed, and the cells were washed RNA was labeled by incubation of cells for 24 h with twice with semifrozen phosphate-buffered saline, 0.1 ,uCi of [14C]uridine per ml. Cultures were inscraped from the dish, and centrifuged at 800 x g for fected and pulse labeled with [3Hluridine, and polyri5 min at 4 C. The cells were disrupted in a Dounce bosomes were prepared on sucrose gradients as homogenizer after suspension in reticulocyte stand- above, except that 10 mM sodium cacodylate, pH ard buffer (10 mM Tris-hydrochloride [pH 7.4], 10 7.4, was substituted for Tris-hydrochloride. PolymM KCl, 1.5 mM MgCl2) plus 0.01% diethylpyrocar- some fractions >808 were pooled and fixed with bonate and swelling for 10 min at 0 C. The nuclear glutaraldehyde as described by Baltimore and fraction was removed by centrifuging at 800 x g for Huang (3) and then centrifuged to equilibrium (100,45 s at 4 C. The nuclei were washed twice with 000 x g for 16 h at 4 C) in 30 to 55% (wt/wt) CsCl reticulocyte standard buffer plus 0.01% diethylpyro- gradients containing 10 mM sodium cacodylate (pH carbonate, and all three supernatants were pooled 7.4), 10 mM NaCl, 1.5 mM MgCl2, and 0.8% Brij-58. and centrifuged at 800 x g and 4 C for 1.5 min. The RNA was prepared from gradient fractions by desaltsupernatant was made 1% in Tween 40 and 0.5% in ing on an agarose column (Bio Gel AO.5M) equilisodium deoxycholate, centrifuged for 10 min at 10,- brated with 10 mM Tris-hydrochloride, pH 7.4, and 1 000 x g and 4 C, layered on a 15 to 55% (wt/vol) mM EDTA and then by Pronase treatment as above. sucrose gradient in a solution of 50 mM Tris-hydroPreparation of poly(A)-containing RNA. RNA chloride (pH 7.5), 25 mM KCl, 5 mM MgCl2, and preparations were dissolved in a solution of 10 mM 0.01% diethylpyrocarbonate, and centrifuged for 15 Tris-hydrochloride (pH 7.6), 0.4 M NaCl, 1 mM h at 30,000 x g and 4 C. The gradient was fraction- EDTA, 10% (vollvol) 0.1% sodium dodecyl sulfate ated and the profile of the absorbancy at 254 nm and passed through a 0.2-ml oligo(dT)-cellulose col(A254) was recorded. The polysomes in the region umn (11). After the eluted RNA was washed with greater than 80S were pooled as the source of the above buffer, it was pooled as poly(A)-minus. mRNA. Care was taken not to include 80S mono- Poly(A)-containing RNA was eluted with a solution somes since influenza virus ribonucleoprotein sedi- of 10 mM Tris-hydrochloride (pH 7.6), 1 mM EDTA, ments around 70S (23). The pooled fractions were and 0.1% sodium dodecyl sulfate. made 0.5% in sodium dodecyl sulfate and precipiRNA-RNA annealing procedures. RNA samples tated with 2 volumes of EtOH. The precipitate was were dissolved in 2x SSC (lx SSC is 0.15 M NaCl dissolved in a solution of 0.1 M Tris-hydrochloride plus 15 mM sodium citrate, pH 7.0). Annealing of (pH 7.5), 50 mM NaCl, 10 mM EDTA, 4 lAg of polyvi- desired mixtures was carried out in 50- or 75-!AI volnyl sulfate per ml, and 0.5% sodium dodecyl sulfate umes in sealed glass ampoules for 18 h at 68 C. and digested with 0.5 mg of Pronase per ml for 30 Samples were recovered in 1.0 ml of 2x SSC. A 0.5min at 37 C (1, 18). After extraction with chloro- ml portion was incubated with 0.5 ml of RNase A form-isoamyl alcohol (24:1, vol/vol), the RNA was (100 ,ug/ml) and RNase T1 (100 U/ml) in 2x SSC for precipitated with 2.5 volumes of EtOH. 30 min at 37 C. The RNase-resistant fraction was Preparation of free and membrane-bound poly- then determined by precipitating the sample and an ribosomes.Two methods were used for the prepara- equivalent undigested sample with 2 ml of 1 N pertion of free and membrane-bound polyribosomes. chloric acid, with 100 jAg of heat-denatured calf thyMethod 1 is a modification of that described above. mus DNA as carrier. The precipitate was collected After the nuclear fraction was washed, the com- on a glass fiber filter, washed with 0.5 N trichloroabined supernatants were centrifuged for 10 min at cetic acid and then EtOH, and dried, and the radioac10,000 x g and 4 C. Free polysomes were prepared tivity was measured in a scintillation spectrometer. from the supernatant on sucrose gradients as above. Preparation of radiolabeled virion RNA. HighThe pellet was dissolved in reticulocyte standard specific-activity 32P-labeled vRNA was prepared as buffer plus 0.1% diethylpyrocarbonate, treated with follows. Chicken embryo cell monolayers in phos-
INFLUENZA VIRUS mRNA
VOL. 16, 1975 phate-free medium 199 were infected with about 0.5 PFU/cell and incubated for 27 h at 37 C in the presence of 200 E&Ci of [32P]P, per ml. The medium was collected, chilled, and centrifuged for 10 min at 10,000 x g and 4 C. The supernatant was centrifuged for 30 min at 120,000 x g and 4 C, and the pellet was suspended in a 0.5-ml solution of 0.1 M NaCl, 10 mM Tris-hydrochloride (pH 7.4), and 1 mM EDTA (NTE), layered on a 15 to 60% (wt/vol) sucrose gradient in NTE (10 ml), and centrifuged for 30 min at 150,000 x g and 4 C. The visible virus band was collected, diluted with 1 volume of water, layered on a 10-ml gradient of potassium tartrate (20 to 45%, wt/vol) in 10 mM Tris-hydrochloride (pH 7.4), and centrifuged for 12 h at 100,000 x g and 4 C. The virus band was diluted with NTE and centrifuged for 30 min at 150,000 x g and 4 C. RNA was extracted by suspending the virus pellet in 0.5 ml of NTE containing 0.5% sodium dodecyl sulfate and 80 itg of tRNA, extracting it twice with NTE-saturated phenol, and then precipitating it with EtOH. 32P-labeled vRNA preparations had specific activities around 1.4 x 106 counts/min per jtg. 3H-labeled vRNA was prepared by the growth of virus in purine- and pyrimidine-free medium 199 supplemented with 1 mCi of [3H]uridine per ml and 1 mCi of [3H]adenosine per ml. 3H-labeled vRNA had a specific activity of 5 x 106 counts/min per Slg.
RESULTS Detection of cRNA in polysomes. All results refer to virus growth at 37 C, except for one set at 31 C described later. Fowl plague virus-infected chick cells were labeled for 30min periods at various times after infection
1437
with [3H]uridine, and polyribosomes were prepared (Fig. 1). RNA was extracted from polysomes and tested for self-annealing in the presence of excess unlabeled vRNA. From 45 min onwards, [3H]RNA complementary to vRNA was detectable (Fig. 2). The rate of appearance of cRNA reached a maximum at 2 to 2.5 h postinfection. At early stages of infection, there was little self-annealing of [3HIRNA, indicating a low content of vRNA in the polysome preparations. However, as infection progressed, increasing amounts of vRNA were present by this criterion. At all stages of infection, high levels of self-annealing (25 to 40%) were observed with [3H]RNA prepared from fractions 80S was analyzed in this study of mRNA. Typically, about four times more 3H label was incorporated into free polysomal RNA than into membrane-bound polysomal RNA. To determine what proportion of 3H-labeled polysomal RNA was cRNA and also to estimate its size, RNA was extracted from free and membrane-bound polysomes prepared from 3Hpulse-labeled chick cells at 2.25 h postinfection and analyzed on sucrose rate zonal density gradients. The 3H-labeled RNA sedimented heterogeneously (range 3S to 20S) with a peak at 13S (Fig. 3). RNA was recovered from each fraction of each gradient by ethanol precipitation and 08
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FIG. 1. Fractionation of polyribosomes. Free polyribosomes were prepared from cells 2.25 h postinfection, sedimented through a sucrose gradient, and colkected into fractions. Stable RNA was labeled with [14C]uridine (16 h at 01 ACilmi) and rapidly labeled RNA with [3H]uridine (30 min at.50 pCi/ml). Symbols: , An, *, 3H counts per minute; 0, 14C counts per minute.
1438
GLASS, McGEOCH, AND BARRY
J. VIROL.
level of annealing to vRNA, whereas the [3H]RNA present in less dense structure shows a high level of self-annealing, with little or no additional annealing to added vRNA. This demonstrates that most of the [3HIcRNA is closely associated with ribosomes, in contrast to vRNA that is present in less dense structures, presumIl A I-
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HOURS POST INFECTION
FIG. 2. Synthesis of polyribosomal virus complementary RNA. Cells were labeled with [3H]uridine for 30 min at various times after infection. Membrane-bound and free polyribosomes were prepared. Self-annealing and cRNA content of the [3HIRNA were measured. Each point represents the end time of a 30-min labeling period. Symbols: 0, percentage of self-annealing of [3H]RNA; *, percentage of 3H in cRNA as determined by annealing to an excess of vRNA.
annealed with an excess of virion RNA. Most of the rapidly labeled RNA was cRNA (Fig. 3) and of a size comparable to the constituents of the fragmented vRNA. We treated isolated polysomes with puromycin in attempts to dissociate and purify mRNA. The [3H]RNA was released and sedimented in sucrose rate gradients at 40S to 60S. A possible explanation for this result is that influenza mRNA in chick cells occurs as a nucleoprotein. Polyribosome-associated messenger ribonucleoproteins have been reported (20). Banding of polysomes and cRNA in CsCl gradients. Cosedimentation ofcRNA with polyribosomes in sucrose gradients does not show conclusively that the cRNA is mRNA. To demonstrate that the [3H]cRNA was closely associated with ribosomes, we fixed polysome preparations with glutaraldehyde and centrifuged them to equilibrium in CsCl density gradients (3) (Fig. 4). In all cases most of the rapidly labeled RNA banded with rRNA (which had been prelabeled with [l4Cluridine) at a density of 1.5 g/ml, as expected for polysomes (3). Some [3HIRNA was present in less dense structures, presumably ribonucleoprotein particles (17), especially in free polysome preparations and at later times. With polysomes prepared 4.5 h after infection and fractionated in CsCl, RNA was extracted from pooled fractions and analyzed for self-annealing and annealing to vRNA (Table 1). For both free and membrane-bound
:i
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FIG. 3. Sedimentation ofpolyribosomal virus complementary RNA in sucrose rate gradients. Free and membrane-bound polyribosomes were prepared from cells 225 h postinfection after 30-min labeling with [3H]uridine 450 ACi/ml). Polysomal RNA was extracted and sedimented on 17 ml of 5 to 20% (wtlvol) sucrose density gradients in 10 mM Tris-hydrochloride (pH 7.4)-0.1 M NaCl-1 mM EDTA-02% sodium dodecyl sulfate buffer at 57,000 x g for 14 h at 22 C. Gradients were collected in 1-ml fractions from the top, and the optical density profile was recorded at 254 nm. 100-pli portions were taken from each fraction to determine trichloroacetic acid-insoluble radioactivity. Each fraction was precipitated with 2 volumes EtOH, collected, and hybridized to vRNA (25 jg/ml). Symbols: A2S4 0, 3H counts per -,
minute hybridized to vRNA;
0,
total 3H counts per
minute. (A) 225 h postinfection, free polysomal RNA; (B) 225 h postinfection, membrane-bound RNA.
INFLUENZA VIRUS mRNA
VOL. 16, 1975
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FIG. 4. Fractionation of polyribosomes in CsCl gradients. Cells were labeled as for Fig. 1. Membranebound and free polyribosomes were prepared at 2.25 and 4.25 h postinfection and fractionated by sedimentation through a sucrose gradient. Fractions ofgreater than 8JS were fixed with glutaraldehyde and centrifuged to equilibrium in CsCL gradients. Symbols: 0, 3H counts per minute; 0, 14C counts per minute; A, density grams per cubic centimeter. (A) 2.25 h postinfection; free polysomes, upper panel; membrane-bound polysomes, lower panel. (B) 4.25 h postinfection; free polysomes, upper panel; membrane-bound polysomes, lower panel. TABLE 1. Density gradient fractionation of rapidly labeled RNA associated with polysomesa Membrane-bound polysomes
Free polysomes Fraction
Polysomes, density 1.5 g/ml Ribonucleoprotein, density 1.34 to 1.39 g/ml
% of total 3H
71.7 28.3
SelfAnnealing annealing to vRNA 5.4 24.3
82.2 19.2
% Of % of
69.2 31.8
SelfAnnealing annealing to vRNA
4.0 18.4
69.9 23.7
a Polyribosomes labeled with [3H]uridine were prepared 4.5 h postinfection on sucrose gradients. Fractions >80s were fixed with glutaraldehyde and centrifuged to equilibrium in CsCl. RNA was extracted from appropriate fractions and tested for self-annealing and annealing to excess vRNA.
ably ribonucleoprotein particles (17). Poly(A) sequences in cRNA. RNA prepared from polysomes 2.25 h postinfection and labeled with [3H]uridine or [3H]adenosine was fractionated on oligo(dT)-cellulose columns (11), and the RNase resistance of the fractions was measured (Table 2). [3H]uridine-labeled cRNA from both membrane-bound and free polysomes gave around 50% binding to oligo(dT)-cellulose, with low levels (2 to 4%) of intrinsic RNase resist-
ance. With [3H]adenosine-labeled RNA, the material binding to oligo(dT)-cellulose had an intrinsic RNase resistance of 22 to 28%. We conclude that about half of the cRNA contained covalently linked poly(A) sequences. Proportion of the virus genome represented in cRNA. The fraction of the influenza virus genome RNA represented in preparations of polysomal RNA was estimated as follows. A series of mixtures was set up in which a con-
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GLASS. McGEOCH, AND BARRY
TABLE 2. Poly(A) content of polyribosomal RNAa % Of Polysomes
% cRNA of 3H
in
cRNA
% 3H RNase rssac
poly(A)
Free [3H]A label [3H]U label
66.7 64.4
54.1 53.4
22.0 2.7
2.3 2.9
Membranebound [3H]A label [3H]U label
70.5 62.3
49.6 41.8
28.0 4.1
2.5 3.7
J. VIROL.
iments with membrane-bound and free polysomal poly(A)-containing RNA from 4.5 h postinfection gave similar results. cRNA present early in infection. RNA was prepared from total polysomes 0.75 h postinfection, which is the earliest time at which cRNA had previously been detected (4). To analyze the expected low levels of cRNA we used 3H-labeled vRNA of specific activity 5 x 106 counts/min per ,ug. A poly(A)-plus fraction of cRNA annealed 92% of the input vRNA. Total polysomal RNA annealed 82%, without reaching a clear plateau at the highest concentration available (data not
shown). After previous work by others (2, 27), we Cells were labeled with [3H]adenosine ([3HIA) or examined, by this method, early events of infec[3Hluridine ([H]U) for 30 min and at 2.25 h postinfection polyribosomes were prepared (method 1) and tion at 31 C. Total polysomal RNA was preRNA was extracted. cRNA content was assayed by pared from cells maintained at 31 C for 1 and 2 annealing samples of [3H]RNA to an excess of h after infection. At both times >90% of the vRNA. Samples were fractionated on oligo(dT)-cellu- genome is represented as cRNA (Fig. 6). lose, and cRNA content and RNase resistance were determined. DISCUSSION Virus mRNA was first detected at 45 min stant amount of 32P-labeled vRNA, usually postinfection, a time when synthesis of total 1,000 counts/min, was hybridized to a large cRNA is just starting (4), so evidently nascent range of concentrations of an unlabeled polysomal RNA preparation. As increasing amounts of cRNA are annealed to a constant amount of vRNA, the final percentage of vRNA found in an RNase-resistant form should represent the proportion of the genome with complementary sequences in polysomal RNA. We first analyzed, by this method, polysomal RNA prepared from cells 2.25 h after infection. RNA from both free and membrane-bound polysomes contained sequences complementary to 0 90 to 100% of the virus genome RNA (Fig. 5A). iO POLYRIBOSOMAL RNA CONCENTRATION mg/ml When free polysomal RNA was fractionated into poly(A)-containing and poly(A)-minus LA 100 B RNA, the poly(A)-minus RNA annealed 100% 80 and the poly(A)-plus RNA annealed 80% of the 4-0-. vRNA (data not shown). These preparations of polysomal RNA were 5Q 60 6 made by method 1 above. We wished to exclude 40 the possibility that the results were affected by cross-contamination of the two polysome 20 classes. Preparations of polysomes were made by method 2, which has been shown to give very 30 40 100 I50 little cross-contamination (Mechler and VasPOLYRIBOSOMAL alli, in press). In this case, only RNA containFIG. 5. Representation of genome sequences in ing poly(A) was used since preparations of [32P]vRNA (1,000 counts/min) was annealed poly(A)-minus RNA made by this method con- cRNA. a range of concentrations of unlabeled polyribosotain vRNA, which could lead to artifactual re- to mal RNA from2 25 h postinfection, and RNase resistsults. As shown in Fig. 5B, both classes anneal ance of the vRNA was determined. (A) Total polyriboabout 80% of the [32P]vRNA. Addition experi- somal RNAs. Symbols: *, total free polysomal RNA; ments in which mixtures of the two polysomal 0, total membrane-bound polysomal RNA. (B) RNAs were annealed to [32P]vRNA gave little Poly(A)-containing RNAs. Symbols: 0, from free or no additivity; 80 to 85% was annealed. Exper- polysomes; 0, from membrane-bound polysomes. a
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FIG. 6. Representation ofgenome sequences in polysomal RNA during infection at 31 C. [3H]vRNA was used, as for Fig. 4. (A) Total polyribosomal RNA from 1 h postinfection at 31 C. (B) Total polyribosomal RNA from 2 h postinfection at 31 C.
cRNA must be rapidly processed into polyribosomal RNA. The maximal rate of appearance of virus mRNA, as measured by 30-min pulses with [3H]uridine, was at 2 to 2.5 h postinfection. These data compare reasonably with previous measurements (24) on the appearance of total cRNA in fowl plague virus-infected cells. At 2 to 2.5 h postinfection, 60 to 70% of the rapidly labeled polysomal RNA is virus cRNA; evidently a large proportion of the synthetic capacity of the cell is turned over to the synthesis of virus-specific products. Both complementary and virus-strand RNA were detectable in polysome fractions from sucrose gradients, the latter increasingly at later times. However, only cRNA behaved as messenger when fixed polysome preparations were fractionated by buoyant density in CsCl. Around 50% of the polysomal cRNA contained poly(A) sequences by the criterion of binding to oligo(dT)-cellulose. There was no evidence of vRNA in the poly(A)-containing fractions. We conclude, in agreement with previous results (12, 22), that virus cRNA behaves as mRNA by the following criteria: (i) rapid labeling with [3H]uridine in polyribosomal fractions of sucrose gradients, (ii) banding with polyribosomes in CsCl density gradients, and (iii) pos-
session of a poly(A) sequence in the molecule. The vRNA found with polysomes in sucrose gradients is probably progeny vRNA, as ribonucleoprotein particles, adventitiously associated with ribosomes (17, 20, 21). We examined the fraction of the genome represented in various polysomal cRNA preparations. Since the influenza virus genome is segmented into RNA pieces, each coding for one protein (10, 28), we are analyzing a population of up to about eight monocistronic mRNA's. At 2.25 h postinfection, both membrane-bound and free polysomal poly(A)-containing RNA contained sequences representing about 80% of the total genome. Mixed annealing experiments showed little or no increase (80 to 85%). Therefore, both polysome classes contain the same 80% of the genome. This result was unexpected. It is considered that the virus glycoproteins hemagglutinin and neuraminidase are synthesized exclusively on membrane-bound ribosomes (9, 14, 16). The genes for these proteins comprise about 30% of the virus genome (10, 28). We, therefore, expected the membranebound polysomal RNA to contain a fraction, 30% of the total, not found in free polysomal RNA. (Such a situation is found with the rhabdovirus vesicular stomatitis virus [7, 131.) The polysome preparations used were made by a method giving minimum cross-contamination of the two classes (Mechler and Vassalli, in press). Similar results were obtained from RNA extracted 4.5 h postinfection. We conclude that there is no absolute segregation of mRNA sequences into the two polysome classes, but our analysis does not discriminate the relative proportions of various mRNA species in a preparation, so it is possible that each polysome class contains distinct ratios of the different mRNA's. This is suggested by Fig. 4A: although low concentrations offree and membrane-bound polysomal RNA give an identical annealing response, much more membrane-bound than free RNA is required to anneal to completion. Shafritz (25) has demonstrated a similar situation in rabbit liver polysomes, where free and membrane-bound fractions each contain mRNA's not normally translated in that class. We were interested in examining the mRNA species detectable at very early times after infection. Analysis of polysomal RNA from 45 min postinfection showed that total polysomal RNA contained sequences complementary to 82% of the virus genome, whereas poly(A)-containing RNA could anneal 92% of the input vRNA. Since, at this time, cRNA synthesis has just started (4), these experiments indicate that, from a very early stage of infection on-
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wards, at 37 C almost all of the virus genome is ity of the various mRNA species, and some transcribed. Skehel (27) has analyzed the syn- sequences could be transcribed much less than thesis of influenza virus-specified polypeptides others early in infection. at early times after infection. He found that at In polysomal RNA obtained at 2.25 h after 31 C, with virus development slowed relative to infection (Fig. 5), we found that poly(A)-contain37 C, some proteins appeared earlier than oth- ing RNA contained sequences complementary ers, suggesting the possibility of selective to around 80% of the genome RNA, whereas mRNA production. Avery and Dimmock (2) in- poly(A)-minus RNA contained sequences comvestigated this possibility by annealing plementary to 90 to 100% of the genome. This [32P]vRNA to total RNA from cells infected result was not due to differences in input cRNA with influenza virus for 2 h at 31 C. They could concentrations. We have no satisfactory expladetect cRNA sequences complementary to only nation for the phenomenon. Conceivably, some 25% of the genome, as opposed to 50% later in poly(A)-minus RNA might not be mRNA but infection, and concluded that a temporal con- might be part of an RNA replication mechatrol of transcription does indeed operate at nism. By the criterion of poly(A) association, at 31 C. In contrast, we found that total polysomal least 80% of the genome codes for mRNA. RNA preparations from 1 and 2 h after infection ACKNOWLEDGMENTS at 31 C contained sequences complementary to thank H. Patmore, R. Farrell, and N. Kitron for We more than 90% of the genome RNA (Fig. 6). technical assistance, Our experimental technique differs from that of of the manuscript. and B. W. J. Mahy for a critical review Avery and Dimmock in two important respects. S. E. G. is a Medical Research Council predoctoral stuFirst, we used radiolabeled vRNA of about 60- dent. This work was supported by grants from the Medical fold higher specific activity, so our experiments Research Council. are more sensitive. Second, we analyzed polyLITERATURE CITED somal RNA containing little or no vRNA, as opposed to total cell RNA, which must contain all 1. Aloni, Y., and G. Attardi. 1971. Symmetrical in vivo transcription of mitochondrial DNA in HeLa cells. of the infecting vRNA. With total cell RNA Proc. Natl. Acad. Sci. U.S.A. 68:1757-1761. preparations from early stages of infection, the 2. Avery, R. J., and N. J. Dimmock. 1975. Temporal conpresence of unlabeled vRNA could lead to artitrol of transcription of influenza virus RNA. Virology 64:409-414. factually low levels of annealing to labeled 3. Baltimore, D., and A. S. Huang. 1968. Isopycnic separavRNA. tion of subcellular components from poliovirus-inOur attempts to detect transcriptional confected and normal HeLa cells. Science 163:572-574. trol were carried out at 31 C (Fig. 6) and were 4. Bean, W. J., and R. W. Simpson. 1973. Primary trandesigned to measure the amount of representascription of the influenza virus genome in permissive cells. Virology 56:646-651. tion of the virus genome in mRNA, rather than D. H. L., J. F. Obijeski, and R. W. Simpson. to measure the quantity of mRNA present. The 5. Bishop, 1971. Transcription of the influenza ribonucleic acid amount of polyribosomal RNA obtained 1 h genome by a virion polymerase. I. Optimal conditions postinfection needed to saturate 3H-labeled for in vitro activity of the ribonucleic acid-dependent ribonucleic acid polymerase. J. Virol. 8:66-73. vRNA (Fig. 6A) suggests that even the most G. 1971. Release, identification and isolation of frequently represented sequences of virus 6. Blobel, messenger RNA from mammalian ribosomes. Proc. mRNA were present in only small amounts at 1 Natl. Acad. Sci. U.S.A. 68:832-835. h after infection at 31 C. We believe, therefore, 7. Both, G. W., S. A. Moyer, and A. K. Banerjee. 1975. Translation and identification of the viral mRNA that at this stage of infection we are looking at species isolated from subcellular fractions of vesicuan intrinsically incomplete process and do not lar stomatitis virus-infected cells. J. Virol. 15:1012wish to place much weight on these results. 1019. However, at 1 h after infection at 31 C, the 8. Chow, N., and R. W. Simpson. 1971. RNA-dependent RNA polymerase activity associated with virions and hybridization curve was biphasic and required subviral particles of myxoviruses. Proc. Natl. Acad. a large amount (12 mg/ml) of polyribosomal Sci. U.S.A. 68:752-756. RNA to saturate the 3H-labeled vRNA (Fig. 9. Compans, R. W. 1973. Influenza virus proteins. II. Asso6A). These data suggest that about half of the ciations with components of the cytoplasm. Virology genome is represented in much greater quan- 10. 51:56-70.R. Compans, W., and P. W. Choppin. 1975. Reproductity than the other half. Nevertheless, this tion of myxoviruses, p. 179-252. In H. Fraenkel-Conof sequence representation gross asymmetry rat and R. R. Wagner (ed.), Comprehensive virology, vol. 4. Plenum Press, New York. had disappeared by 2 h postinfection at 31 C. We conclude that influenza virus does not ex- 11. Edmonds, M., and M. G. Caramela. 1969. The isolation and characterization of adenosine-monophosphatehibit a temporal control of transcription, where rich polynucleotides synthesized by Ehrlich ascites "temporal control" means that some sequences cells. J. Biol. Chem. 244:1314-1324. are not transcribed until later times. However, 12. Etkind, P. R., and R. M. Krug. 1974. Influenza viral messenger RNA. Virology 62:38-45. our technique does not demonstrate equimolar-
VOL. 16, 1975 13. Grubman, M. J., E. Ehrenfeld, and D. F. Summers. 1974. In vitro synthesis of proteins by membranebound polyribosomes from vesicular stomatitis virusinfected HeLa cells. J. Virol. 14:560-571. 14. Hay, A. J. 1974. Studies on the formation of the influenza virus envelope. Virology 60:398-418. 15. Hulse, J. L., and F. 0. Wettstein. 1972. Two separable cytoplasmic pools of native ribosomal subunits in chick embryo tissue culture cells. Biochim. Biophys. Acta 269:265-275. 16. Klenk, H. D., W. W6llert, R. Rott, and C. Scholtissek. 1974. Association of influenza virus proteins with cytoplasmic fractions. Virology 57:28-41. 17. Krug, R. M. 1972. Cytoplasmic and nucleoplasmic viral RNPs in influenza virus-infected MDCK cells. Virology 50: 103-113. 18. Miller, L., and G. Knowland. 1970. Reduction of ribosomal RNA synthesis and ribosomal RNA genes in a mutant of Xenopus laevis which organizes only a partial nucleolus. II. The number of ribosomal RNA genes in animals of different nucleolar types. J. Mol. Biol. 53:329-338. 19. Penhoet, E., H. Miller, M. Doyle, and S. Blatti. 1971. RNA-dependent RNA polymerase activity in influenza virions. Proc. Natl. Acad. Sci. U.S.A. 68:13691371. 20. Penman, S., C. Vesco, and M. Penman. 1968. Localization and kinetics of formation of nuclear heterodisperse RNA, cytoplasmic heterodisperse RNA and po-
INFLUENZA VIRUS mRNA
21.
22.
23.
24.
25.
26.
27.
28.
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lyribosome-associated messenger RNA in HeLa cells. J. Mol. Biol. 34:49-69. Perry, R. P., and D. E. Kelley. 1968. Messenger RNAprotein complexes and newly synthesized ribosomal subunits: analysis of free particles and components of polyribosomes. J. Mol. Biol. 35:37-59. Pons, M. W. 1971. Isolation of influenza virus ribonucleoprotein from infected cells. Demonstration of the presence of negative-stranded RNA in viral RNP. Virology 46:149-160. Pons, M. W. Studies on the replication of influenza virus RNA. Virology 47:823-832. Scholtissek, C., and R. Rott. 1970. Synthesis in vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40:989-996. Shafritz, D. A. 1974. Evidence for nontranslated messenger ribonucleic acid in membrane-bound and free polysomes of rabbit liver. J. Biol. Chem. 249:89-93. Siegert, W., G. Bauer, and P. H. Hofschneider. 1973. Direct evidence for message activity of influenza virion RNA. Proc. Natl. Acad. Sci. U.S.A. 70:29602963. Skehel, J. J. 1973. Early polypeptide synthesis in influenza virus-infected cells. Virology 56:394-399. White, D. 0. 1974. Influenza viral proteins: identification and synthesis. Curr. Top. Microbiol. Immunol. 63:1-48.
