VIROLOGY 83, 337-355 (1977)

Transcription A. J. HAY,’ Division

of the Influenza

B. LOMNICZI,2

of Virology,

National

Institute

Virus Genome

A. R. BELLAMY,3

AND

J. J. SKEHEL

for Medical Research, Mill Hill London NW? 1AA

Accepted July 28,1977 Analyses of the synthesis of virus-specific RNA in fowl plague virus-infected chick embryo cells are reported. The genome of this strain of influenza virus appears to consist of 10 single-stranded RNAs with molecular weights ranging from approximately lo5 to 106. The synthesis of both virion RNA and complementary RNA occurs mainly at early times during infection. The complementary RNAs are of two types which are distinguished by a number of properties, and each population, which is made up of molecules complementary to all 10 genome RNAs, clearly serves a different function. The polyadenylated cRNAs which appear to be incomplete transcripts of each genome RNA molecule are exclusively associated with polysomes and therefore comprise the virus mRNA. The synthesis of these molecules is controlled throughout infection with respect to both the amount of each transcript produced and the time at which it is produced in maximum amount. The unpolyadenylated cRNAs on the other hand are complete transcripts of each genome RNA and are produced in similar relative amounts during 3 hr of infection; it is suggested that these molecules serve as template for replicating the virus genome. It is also apparent from these investigations that the two types of cRNA are synthesised by the same transcriptase but that the production of unpolyadenylated complete transcripts is dependent upon the synthesis of certain other viral protein(s). INTRODUCTION

From a number of reports it is apparent that the genomes of influenza viruses are made up of between 8 and 10 distinct single-stranded RNA molecules with molecular weights of between 2 x lo5 and lo6 (Lewandowski et al., 1971; Pons, 1976; Palese and Schulman, 1976; McGeoch et al., 1976). The virus-specific messenger RNA consists of molecules complementary in sequence to these genome RNAs (Pons, 1972; Etkind and Krug, 1974; Glass et al., 1975; Stephenson et al., 1977). There is, however, little detailed information concerning the transcription of the influenza virus genome during infection and in this communication two related questions are I Author to whom reprint requests should be addressed. * Present address: Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest, Hungary. 3 Present address: Department of Cell Biology, University of Auckland, Auckland, New Zealand.

considered: (i) Is the synthesis of the virusspecific messenger RNAs regulated during infection so as to control the synthesis of virus-specified polypeptides (Skehel, 1972, 1973)? (ii) Since virus-specific complementary RNA has to serve two functions, namely, as messenger RNA and as template for replicating the genome, do the same molecules perform these two functions or are they carried out by separate populations of complementary RNA? Results are presented which indicate that the genome of fowl plague virus contains 10 RNA molecules and that during infection the transcription of these RNAs is a highly regulated process which produces controlled amounts of each mRNA and a second population of complementary RNAs which in all probability serves as template in genome replication. MATERIALS

AND

METHODS

Viruses and cells. The Restock strain of fowl plague virus was grown and purified 337

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN

0042-6622

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ET AL.

as described previously (Hay, 1974). Primary CEF4 cells were prepared as described by Porter-field (1960) and incubated in Gey’s medium containing 10% calf serum. Infection of cells and radioactive labelling. CEF monolayers (5 x lo6 or 5 x lo7 cells/culture) were infected with fowl plague virus at an added multiplicity of 100 PFU per cell for 30 min at Zo”, washed in Tris-buffered twice, and incubated Gey’s medium. Cells were labelled either containing by incubating in medium [3H]uridine (100 &i/ml) or in phosphatefree medium containing [32Plorthophosphate (1 mCi/ml). Cell fractionation. This was carried out essentially as described by Hay (1974). Polysome isolation was carried out using either of two buffers: 1O-2 M NaCl, lop2 M Tris-HCl, pH 7.5, 10e3 M MgCl,; or 2 x 10-l M KCl, 10e2 M Tris-HCl, pH 7.5, 10e2 M MgCl,. The cytoplasmic extract was centrifuged on a 15-40% sucrose gradient containing the appropriate buffer at 170,000 g for 3.5 hr using an SW 41 rotor. The gradient was scanned continuously at 260 nm during collection. RNA extraction. Cell monolayers were washed three times with cold saline and dissolved in 0.5% SDS, 10e2 M sodium acetate, pH 5. The solution was extracted twice with an equal volume of water-saturated phenol and the RNA was precipitated from the aqueous phase by the addition of 2 vol of ethanol. Pelleted fractions were treated similarly. To cell homogenates, cytoplasmic extracts, and gradient fractions was added 0.5% SDS, 5 x lop3 M EDTA, and 5 x lop2 M sodium acetate, pH 5, and the RNA was extracted as above. RNA extraction using a phenolchloroform (1:l) mixture was found to yield similar results. The precipitated RNA was 4 The following abbreviations are used: PFU, plaque-forming units; CEF, chick embryo fibroblast; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulphate; CTAB, cetyltrimethylammonium bromide; BIS, NJV’-methylene-bisacrylamide; TEMED, NJVJV’,?V’-tetramethylenediamine; TCA, trichloroacetic acid; mRNA, messenger RNA; cRNA, complementary RNA; vRNA, viral RNA; RNP, ribonucleoprotein.

washed as follows: It was dissolved in lo-’ M sodium acetate, pH 5, containing 2 x lo-* M sodium pyrophosphate and reprecipitated by the addition of 0.2% cetyltrimethylammonium bromide (CTAB). The precipitate was washed with lo-’ M sodium acetate, pH 5, 5 x lop2 M NaCl, containing 0.2% CTAB and then twice with 70% ethanol containing 10-l M NaCl. To a suspension of purified virus in 10-l M NaCl, 10e2 M Tris-HCl, pH 7.5, was added 1% SDS, 5 x 10m2M sodium acetate, pH 5, 1O-3 M EDTA and the solution was extracted three times with an equal volume of phenol and three times with an equal volume of ether. The precipitated RNA was washed three times with 70% ethanol containing 3 x lop2 M NaCl. Isolation of polyadenylated RNA. RNA dissolved in 0.5 M LiCl, 0.5% lithium dodecyl sulphate, 10m2M Tris-HCl, pH 7.5, was applied to a column of oligo(dTl-cellulose equilibrated with the same buffer. The eluate was reapplied to the column and the column was washed with the buffer until all unbound radioactivity had been removed. The bound RNA was then eluted with lop2 M Tris-HCl, pH 7.5 and the RNA in both fractions was recovered by precipitation with ethanol. An alternative procedure used to separate polyadenylated double-stranded RNA from unpolyadenylated double-stranded molecules formed during annealing experiments involved precipitating the former in the presence of 2 M LiCl at 4” for 12 hr. Under these conditions the completely double-stranded RNA did not precipitate and was recovered by ethanol precipitation. Similar results were obtained when the polyadenylated and unpolyadenylated RNAs were separated using oligo(dT)-cellulose before hybridisation. RNA-RNA hybridisation. This was carried out essentially as described by Ito and Joklik (1972). RNA mixtures in 10m2 M NaCl, 1O-2 M Tris-HCl, pH 7.5, 10-l M EDTA were denatured by adding 9 vol of dimethyl sulphoxide and incubating at 45” for 30 min. NaCl, Tris-HCl, pH 7.5, and EDTA were added to give final concentrations of 3 x 10p2, 10p2, and 1.5 x 10m3M, respectively, the dimethyl sulphoxide con-

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centration was reduced to 63%, and incubation was continued at 37” for 20 hr. The RNA was precipitated and washed twice with 70% ethanol containing 2 x 10e2M NaCl. Quantitative analyses of virus-specific RNA. Measurements of virus-specific

RNA

SYNTHESIS

339

in 10-l M NaCl, lo-* M sodium acetate, pH 4.5, 5 x 10e4M ZnS04 was incubated at 37” for 4 hr in the presence of 1000 U/ml of nuclease S,. The RNA was reprecipitated with ethanol, washed, and dissolved in lo-* M Tris-acetate,, pH 7.8, 7 M urea, 5 x 1O-3 M EDTA. Other ribonucleases tested including A, T1, and T, were found to be less satisfactory.

complementary RNA (cRNA) were made by annealing 3H-labelled RNA extracted from infected cells in the presence of an Gel electrophoresis and autoradiograexcess of purified viral RNA. Conditions phy . Samples of viral RNA dissolved in 7 of “viral RNA excess” were determined by M urea, 5 x 10m3M EDTA were analysed annealing a mixture of 3H-labelled viral by electrophoresis on either: (i) polyacrylRNA and unlabelled RNA isolated from amide slab gels (15 cm) containing 3% cells at 4 hr after infection in the presence acrylamide, 0.15% N, N’-methylene-bisof increasing amounts of unlabelled viral acrylamide (BIS), 0.4% N,N,N’,N’-tetraRNA. Twenty micrograms of unlabelled methylethylenediamine (TEMED), 2 x viral RNA in the presence of RNA from 2 10e3M EDTA, 2.5 x lo-* M sodium citrate, x 10’ cells reduced the annealing of the pH 3.5, and 1 mg/ml of ammonium persul3H-labelled viral RNA to less than 2% of phate at 70 V for 16 hr; or (ii) agarose gels the control and these conditions were cho- (20 cm) containing 4% agarose, 7 M urea, sen for routine analyses. 2 x lo-* M EDTA, 4 x lo-* M TrisTo determine total virus-specific RNA, acetate, at 50 V for 20 hr. 3H-labelled infected cell RNA was denaSamples containing double-stranded tured and annealed in the presence of a RNA dissolved in 7 M urea, 5 x lo-* M similar excess of unlabelled “double- EDTA, lo-* M Tris-acetate, pH 7.8, were stranded” RNA. This RNA was synthe- analysed by electrophoresis on either: (i) sised in vitro using the virion-associated polyacrylamide slab gels containing 4% transcriptase (manuscript in preparation) acrylamide, 0.2% BIS, 0.4% TEMED, 0.1% and consisted of double-stranded and sin- SDS, lo-* M EDTA, 4 x lo-* M Trisgle-stranded molecules. The ratio of virion acetate, pH 7.8, and 1 mg/ml of ammoRNA:complementary RNA in this prep- nium persulphate at 80 V for 16 hr; or (ii) aration was approximately 2:l and the polyacrylamide slab gels containing 7.5, RNase-resistance data were corrected ac- 10, or 12% acrylamide and, respectively, cordingly. In addition all data were cor- 0.2, 0.26, or 0.31% BIS and 0.4% TEMED, rected with respect to uninfected controls 6 M urea, 0.1% SDS, lo-* M EDTA, 4 x lo-* M Tris-acetate, pH 7.8, and 1 mglml and with respect to efficiencies of hybridisation estimated using labelled vRNA and of ammonium persulphate at 50 V for 40cRNA (synthesized in vitro). The amount 50 hr. Gels containing 32P-labelled samples of labelled viral RNA was then determined by the difference between the values for were processed for autoradiography as described by Russell and Skehel (1972) and total and complementary RNAs. The amount of radioactive RNA which gels containing 3H-labelled samples were annealed under these conditions was de- processed for fluorography as described by termined by incubating samples of the Bonner and Laskey (1974) and exposed to annealed RNA dissolved in 2 x 10-l M preexposed film (Laskey and Mills, 1975). Materials. [5,6-3HlUridine (49 Ci/mmol) NaCl, lo-* M Tris-HCl, pH 7.5, in the presence of 10 pg/ml of ribonuclease A at and [32Plorthophosphate (carrier-free) 37” for 30 min and measuring the amount were obtained from The Radiochemical Centre, Amersham. Nuclease S1and cycloof ribonuclease-resistant radioactivity. Before polyacrylamide-gel analyses of heximide were obtained from the Sigma the labelled double-stranded RNAs formed Chemical Co. Oligo(dT)-cellulose was obduring hybridisation, the RNA dissolved tained from Collaborative Research Inc.

HAY ET AL.

340

The constituents used to make polyacrylamide gels were obtained from Eastman Kodak Ltd. and purified as described previously (Hay et al., 1967).

A

B

RESULTS

Analysis

of the Virus Genome

Analyses of virion RNA by electrophoresis on polyacrylamide or agarose gels in the presence of urea have indicated that fowl plague virus contains 10 single-strand RNA molecules (Fig. 1). Approximate molecular weights of these RNAs, estimated using ribosomal RNAs as markers, range from lo5 to 106. Similar estimates of approximately lo5 daltons for the sizes of RNAs 9 and 10 were obtained from electrophoresis on polyacrylamide gels containing 99% formamide, including 7, 5, and 4 S RNAs as markers. Estimates of the sizes of the RNAs obtained from a comparison of the electrophoretic mobility of the corresponding double-stranded RNA molecules (see below) with the mobility of reovirus double-stranded RNAs were somewhat lower. In view of the observations of Bozarth and Harley (19761, however, it is likely that estimates of the sizes of the smaller RNA molecules and in particular RNAs 9 and 10 by this method are rather inaccurate. That the order of migration of the single-stranded RNA molecules 1-8 was the same as that of the corresponding double-stranded molecules was confirmed by ribonuclease T, oligonucleotide fingerprint analyses (unpublished results). Virus-Specific

1,23-

78-

9IO-

RNA Synthesis

Virus-specific RNA was detected, as described in Materials and Methods, by annealing 3H-labelled RNA isolated from fowl plague virus-infected cells in the presence of an excess of either (i) unlabelled virion RNA or (ii) unlabelled doublestranded virus RNA, synthesised in vitro. Samples analysed using the first method allowed estimates of the amount of RNA complementary to the genome (cRNA) and, using the second, of the total amount of virus-specific RNA. The amount of virion RNA (vRNA) was thus estimated by the difference between these two values.

FIG. 1. Analysis of virion RNA. (A) Unlabelled viral RNA was analysed on a 3% polyacrylamide gel, as described in Materials and Methods. (B) lz51labelled viral RNA, prepared as described by Abraham et al. (in preparation), was analysed on a 4% agarose gel as described in Materials and Methods.

The Synthesis

of Virion RNA

The synthesis 1.5 to 2 hr after creasing steadily mum 3 hr later differ somewhat

of vRNA was maximal infection, thereafter deto around 6% of maxi(Fig. 2B). These results from those reported by

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SYNTHESIS

others (Scholtissek and Rott, 1970; Taylor et al., 1977) who observed that vRNA synthesis was predominant at later times during infection and, although in the latter instance the difference may be explicable in terms of the different virus-cell system studied, the reason for the difference between these results and those of the former report is unclear. Experiments to determine when the genome of released virus particles was synthesised also indicated that genome RNA synthesis was maximal 1.5-2 hr after infection and that the RNA components of 90% of the virus particles produced throughout infection were synthesised by 4 hr (Fig. 2A), i.e., before significant amounts of progeny virus had been released (Hay, 1974). Analyses of the RNA of virus particles released from cells labelled at different times after infection, as described in Fig. 2A, showed that the relative labelling of the individual RNAs was invariant throughout infection and, therefore, that the different genome RNA molecules were synthesised at the same time during infection.

-B

CPM X10”

The Synthesis (cRNA)

HOURS Fro. 2. Kinetics of the synthesis of virus-specific RNA in fowl plague virus-infected CEF cells. (A) Incorporation’of RNA into released virus particles. Experiment 1 (O-01: At 1, 1.5, 2, 3, and 4 hr after infection 10 &i/ml of [%H]uridine was added to duplicate cultures of CEF cells (6 x lo5 cells/ culture) and medium was harvested at 12 hr after infection. Experiment 2, (O-----O): At 1, 1.5, 2, 2.75, 3.5, and 4.5 hr after infection 40 &i/O.2 ml of 18H1uridine was added to duplicate cultures of CEF cells (5 x lo8 cells/culture) and, after 20 min, cells were washed three times and incubated in medium containing 10m3M uridine. Culture fluid was harvested at 12 hr after infection. Virus was purified from the culture medium as described previously (Hay, 1974) and the total radioactivity in the released virus was determined. (B) At different times after infection, duplicate cultures of CEF cells (5 x lad cells/culture) were incubated for 30 min in medium containing 100 @i/ml of PHluridine. RNA was extracted, aliquots were denatured and an-

of Complementary

RNA

The synthesis of cRNA was maximal at 1.5 to 2 hr after infection, as was that of vRNA, and continued at a reduced rate throughout infection (Fig. 2B). Virus-specific complementary RNA was found to be composed of two distinct types of molecules, those which have poly(A) sequences at their 3’-termini (Etkind and Krug, 1974) and those which lack added poly(A), populations readily separated by affinity chromatography using oligo(dT)-cellulose. The relative rates of production of these two types of cRNA varied throughout infection, the rate of formation of polyadenylated cRNA always exceeding that of the unpolyadenylated molecules although nealed, as described in Materials and Methods, in the presence of an excess of either unlabelled virion RNA or unlabelled “double-stranded” RNA, and the radioactivities in cRNA (0-O) and vRNA (O-----O) were determined. (C) The relative radioactivities in polyadenylated cRNA (0-O) and unpolyadenylated cRNA (O-----O) obtained from the experiment described in Fig. 4.

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HAY ET AL

over the first hour of infection they were produced in similar amounts (Fig. 20. The time at which synthesis was maximal also differed since the rate of production of unpolyadenylated cRNA reached a maximum somewhat earlier, at approximately l-l.5 hr, than the polyadenylated cRNA which was produced maximally at about 2 hr after infection. Moreover, the synthesis of unpolyadenylated cRNA was more drastically curtailed later in infection. These results suggest, therefore, that the synthesis of these two types of cRNA is separately controlled and this suggestion was reinforced by the results of the following analyses of the rates of synthesis of the individual RNA molecules of the two populations. The Composition

of cRNA

The labelled hybrid molecules formed by annealing 3H-labelled infected cell RNA in the presence of an excess of virion RNA were treated with nuclease S, to remove any unhybridised sequences such as poly(A), and the double-stranded RNAs were then analysed by electrophoresis on polyacrylamide gels. S, nuclease degrades specifically single-stranded DNA or RNA (Shishido and Ando, 1972), and the 5’and 3’-termini of reovirus double-stranded RNA have been shown to be completely resistant to its action (Muthukrishnan and Shatkin, 1975). That the efficiencies of hybridisation of the different RNA molecules were similar and hence that the pattern of labelled double-stranded RNA molecules is a true reflection of the composition of labelled cRNA was confirmed in two ways. (i) Labelled virion RNA was annealed in the presence of an excess of unlabelled cRNA isolated from cells infected in the presence of cycloheximide (see below). The relative radioactivities of the different virion RNAs and the corresponding hybrid molecules were similar. (ii) Complementary RNA synthesised in vitro by the virion-associated RNA transcriptase was isolated as double-stranded molecules and following denaturation and reannealing the composition of this RNA was unchanged (manuscript in preparation). The double-stranded RNA molecules

were analysed by electrophoresis either on 4% polyacrylamide gels in the absence of urea, conditions which resolved all 10 double-stranded RNAs, or on 7.5% polyacrylamide gels containing urea, conditions which did not resolve doublestranded RNAs 2 and 3 but facilitated the detection of bands 9 and 10. Transcripts of all 10 genome RNAs were detected during infection and were present in both polyadenylated and unpolyadenylated RNA populations (Fig. 3A). Although transcripts 9 and 10 and in particular the unpolyadenylated transcript 10 were detected less readily than the other transcripts due to their low radioactivity relative to the background in this region of the gel, they have consistently been observed specifically in virus-infected cells (see Figs. 4 and 8) as well as in infected cells treated with actinomycin D indicating that these are indeed transcripts of the virus genome. That the doublestranded molecules from the two different types of cRNA migrate in the same order and correspond as indicated has been confirmed by ribonuclease T, oligonucleotide mapping (unpublished results). The double-stranded RNAs derived from polyadenylated cRNA have a higher electrophoretic mobility than the corresponding molecules derived from unpolyadenylated cRNA (see also Figs. 7 and 81, and this appears to be because the polyadenylated cRNAs represent incomplete transcripts of the viral RNAs in contrast to the unpolyadenylated molecules which appear to be complete transcripts. This conclusion is based on the following observations: (i) Double-stranded RNA molecules derived from unpolyadenylated cRNA migrated as discrete bands during electrophoresis on polyacrylamide gels prepared using high concentrations of acrylamide, e.g., 12%, and their migration was unaffected by prior treatment with nuclease S,, suggesting that they are completely double-stranded. (ii) Hybrid molecules formed between vRNA and polyadenylated cRNA and not subjected to nuclease treatment failed to enter such gels. However, cRNA which is synthesised in vitro by the virion-associated RNA tran-

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-12,3

2,3& :=

z#

6-

-6

7-

-7

8-

-8

-9 -10

4%

7.5%

FIG. 3A. Compositions of unpolyadenylated cRNA (A) and polyadenylated cRNA (B) isolated from fowl plague virus-infected CEF cells. Infected cells (5 x 10’ cells/culture) were incubated for 3 hr in medium containing 100 @i/ml of 32P. The RNA was extracted, denatured, annealed in the presence of excess unlabelled virion RNA (100 gg), and fractionated using oligo(dT)-cellulose. The RNA in each fraction was treated with nuclease S,, and the double-stranded RNAs were separated by electrophoresis either on a 4% polyacrylamide gel at 90 V for 18 hr or on a 7.5% polyacrylamide gel at 50 V for 40 hr, and detected by autoradiography.

scriptase and which does not contain poly(A) produces double-stranded molecules equivalent in size to those obtained from polyadenylated cRNA. Without prior these doublenuclease S, treatment stranded RNAs migrate more slowly as

diffuse bands indicating the presence of single-stranded regions presumably of the vRNA strand (manuscript in preparation). Furthermore preliminary data from sequence analyses supports this conclusion. Size estimates of the double-stranded mol-

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HAY ET AL.

i

i

i 1 ‘i Si

FIG. 3B. Microdensitometer tracings of autoradiograms of the 4% polyacrylamide gel shown in Fig. 3A. (A) Unpolyadenylated cRNA; (B) polyadenylated cRNA. The direction of migration was from right to left.

ecules indicated a difference of approximately lO,OOO-15,000 daltons (30-40 bases) between the sizes of the two types of transcript.

The Synthesis of Polyadenylated cRNAs As seen from the results presented in Figs. 3A and 3B the various polyadenylated cRNA molecules were synthesised in quite different amounts during 3 hr of infection. Thus, for example, transcript 5 was produced in the greatest amount and at around 10 times the level of transcript 2. From analyses of the cRNA synthesised during 30 min periods at different times throughout infection (Figs. 4 and 5A-50 the following conclusions were drawn concerning the synthesis of polyadenylated cRNA.

(1) The various polyadenylated cRNAs were synthesised in different relative amounts at different times during infection. Thus, immediately after infection transcripts l-8 were almost equally labelled. Between 30 and 90 min after infection transcripts 5 and 8 were synthesised preferentially, and at times later than 2.5 hr after infection transcripts 4, 5, and 7 were made in greatest amount. (2) Certain polyadenylated cRNAs, which included transcripts 1-3, were synthesised in relatively small amounts at all times throughout infection. (3) The maximum rate at which the various polyadenylated cRNAs were produced occurred at different times during infection and from their time course of synthesis the transcripts could be divided into four categories. (i) Transcript 8: Synthesis increased sharply after 1 hr and reached a maximum around 1.5 hr, thereafter decreasing rapidly to approximately 10% of maximum by 3.5 hr. (ii) Transcripts 1, 2, 3, and 5: Synthesis was 50% of maximum around 1.25 hr, reached a peak around 2 hr, and was still greater than 20% of maximum at 4.5 hr. (iii) Transcripts 4, 6, 7, and 10: Synthesis was 50% of maximum around 1.75 hr, reached a peak between 2 and 2.5 hr, and was still approximately 30% of maximum at 4.5 hr. (iv) Transcript 9: Synthesis increased steadily throughout infection and persisted at later times. These results indicate, therefore, that synthesis of polyadenylated cRNAs is strictly controlled with respect to both the amount of each transcript produced and the time at which the transcripts are produced in greatest amount. In cells infected in the presence of cycloheximide under conditions which completely block protein synthesis the only virus-specific transcription is catalysed by the enzyme associated with infecting virus particles; this is referred to as primary transcription. Under these conditions transcripts of all 10 genome RNAs were detected (Figs. 4 and 5A). Furthermore, the pattern of synthesis of the polyadenylated cRNAs was similar to that observed immediately aRer a normal infec-

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6 71c

RNA

tion although the amount of synthesis was somewhat greater (Figs. 5B and 7) and remained unaltered over at least 5 hr of infection. The Synthesis

2,3L 4567-8--

ix

.-

9-

--““-

“F

$8

IOFIG. 4. Time course of the synthesis of polyadenylated cRNAs in fowl plague virus-infected CEF cells. Infected cells (5 x 10’ cells/culture) were incubated in medium containing 100 $i of [SH]uridine/ml/culture for 30-min periods from 0 hr (l), 0.5 hr (2), 1 hr (3), 1.5 hr (4), 2 hr (W, 3 hr (6), and 4.5 hr (7) after infection. Uninfected (U) cells and cells infected and incubated in the presence of cycloheximide, as described in Fig. 7 (lc), were labelled similarly at time (1). Cells were washed, suspended in lo-* M NaCl, 10m2M Tris-HCl, pH 7.5, 10ds M MgClz and broken by Dounce homogenieation. One-half of the homogenate was fractionated as described in Fig. 8. RNA extracted from the other half was denatured, annealed in the presence

345

SYNTHESIS

of Unpolyadenylated

cRNAs

In contrast to the disproportionate synthesis of the different polyadenylated cRNA molecules the unpolyadenylated cRNAs l-8 were present in similar amounts at 3 hr after infection (Figs. 3A and 3B). On the other hand the unpolyadenylated transcript 9 was present in substantially greater amounts while transcript 10 was barely detectable (Fig. 3A). Furthermore, the relative rates at which the different unpolyadenylated cRNAs were made remained fairly constant over 2 hr of infection while at later times the pattern of synthesis reflected to some extent the synthesis of polyadenylated cRNAs; in particular the synthesis of transcript 8 was markedly curtailed, and transcript 9 continued to be made in increasing amounts up to at least 5 hr (Fig. 6). There was no detectable synthesis of unpolyadenylated cRNA in cells at any time following infection in the presence of cycloheximide as described above (Fig. 71, indicating that protein synthesis is required for the formation of unpolyadenylated cRNA molecules. Two further observations were apparent from the results of these and other experiments and are particularly relevant to questions relating to the synthesis of unpolyadenylated cRNA as discussed later. The amount of polyadenylated cRNA synthesised in the presence of cycloheximide was, on the one hand, similar to the sum of polyadenylated and unpolyadenylated cRNAs synthesised immediately after a normal infection and, on the other hand, of an excess of unlabelled virion RNA, and fractionated using oligo(dT)-cellulose into polyadenylated and unpolyadenylated molecules; the RNA in each fraction was treated with nuclease S,. The doublestranded RNAs from the polyadenylated RNA fraction were separated by electrophoresis on 7.5% polyacrylamide gels and detected by fluorography. The photograph of the lower end of the gel is from a 15 day exposure while that of the rest of the gel is from a B-day exposure.

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ET AL.

1 2 3 4*

Transcription of the influenza virus genome.

VIROLOGY 83, 337-355 (1977) Transcription A. J. HAY,’ Division of the Influenza B. LOMNICZI,2 of Virology, National Institute Virus Genome A...
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