VIROLOGY 64,409-414
(1975)
Temporal
Control
of Transcription
of Influenza
Virus RNA
ROGER J. AVERY AND NIGEL J. DIMMOCK Department
of Biological
Sciences, University Accepted
of Warwick, Coventry CV4 7AL, England
November
20, 1974
Only part of the single-stranded RNA genome of influenza virus is transcribed early in infection. The appearance of the RNA synthesised later in infection is prevented by actinomycin D but not by cycloheximide. INTRODUCTION
In many cases it has been shown that the expression of the information present in a viral genome occurs in a controlled fashion. Thus the successive and orderly synthesis of distinct classes of RNA is known in both phage and animal virus-infected cells. Influenza virus possesses a segmented single-stranded genome (Duesberg, 1968; Pons and Hirst, 1968; Skehel, 1971) which is transcribed into complementary RNA (cRNA) in infected cells (Bean and Simpson, 1974; Stephenson and Dimmock, 1975). This cRNA has been implicated as influenza virus messenger RNA (Pons, 1972; Kingsbury and Webster, 1973). Virus particles contain a virion polymerase that in vitro makes RNA complementary to the genome (Chow and Simpson, 1971), and this may well be the enzyme responsible for at least some transcription in uiuo. In vitro studies have shown that this enzyme can copy all or nearly all of the viral genome into cRNA (Bishop et al., 1972). Thus influenza virus might be expected to express all its genetic information immediately on infection. However, Skehel (1973) has found that early in infection with influenza virus only three virus-specific proteins are synthesized but at later times all the viral proteins are present. This clearly indicates that influenza virus does exert temporal control over the expression of its genetic information. The important question which then remains to be asked is at what stage does this control occur? If all 409 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
influenza virus messenger RNA’s are made throughout the growth cycle, then control must be exerted on the translation of these RNA’s. On the other hand, transcription may itself be controlled in uiuo. In order to resolve this problem we have examined the cRNA synthesised early in infection by influenza virus and determined what proportion of the viral genome is represented in these transcripts at various times. MATERIALS
AND METHODS
Virus. A recombinant influenza virus strain between A/FPV/Dutch/27 (Hav 1 Neq 1) and AO/BEL/42 (HON 1) designated FPV/BEL was grown in embryonated chicken’s eggs (Stephenson and Dimmock, 1975). Growth of 32P-labelled virus in deem bryonated eggs. The method for prepar-
ing infectious virus with a specific activity of about 80,000 cpm/pg of RNA has been described (Stephenson and Dimmock, 1975). Before extracting the RNA the virus was dialysed against 0.1 x SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Growth
of virus
in chick embryo cells.
Chick embryo cells, 7.5 x lo7 per 14-cm petri dish (Morser et al., 1973) were infected with virus at an input multiplicity of 10 PFU/cell, and virus and was allowed to adsorb for 45 min at room temperature. The cells were washed with maintenance medium at 31” and then incubated in 15
410
AVERY
AND DIMMOCK
ml of this medium at the same temperature. Maintenance medium consisted of 2% calf serum in Medium 199 buffered with bicarbonate. At 2 or 6 hr postinfection the medium was removed and the cells were washed twice with phosharvested, phate-buffered saline and resuspended in 0.01 M NaCl, 0.001 M EDTA and 0.01 M sodium acetate, pH 5.2. When labelled RNA was required, maintenance medium was replaced by bicarbonate-buffered Earle’s saline containing 2% dialysed calf serum, and 15 PCi of [3H]uridine per ml. Preparation of RNA. RNA was prepared from virus and infected cells as described by Avery (1974). Hybridization. All RNA samples to be hybridized were dissolved in 0.04 x SSC. Radioactively labelled virus-particle RNA and unlabeled RNA extracted from infected cells were mixed and denatured by heating at 98” for 4 min and then rapidly brought to 6 x SSC and 65”. Incubation was carried on for 4 hr at this temperature. Subsequent procedures have been described previously (Avery, 1974)) except that T, ribonuclease was used at a concentration of 850 units/ml. Radioactively labelled virion RNA showed between 0 and 0.5% ribonuclease resistance, and this was not increased on selfannealing. Measurement of protein synthesis in the presence of cycloheximide. Chick embryo cells (9 x lo6 cells per 5-cm dish) were pretreated for 1 hr with various amounts of cycloheximide at 37”. Virus was adsorbed in the presence of the drug at room temperature. Infected cells were placed at 31’ and 2.5 PCi of a ‘“C-labelled amino acid mixture (57 mCi/mAtom carbon, The Radiochemical Centre, Amersham) was added per plate (zero time), Cells were precipitated with ice-cold 5% TCA at 1 or 6 hr postinfection, collected on cellulose acetate filters, and washed three times with 5% TCA and once with ether. Filters were dried and counted. RESULTS
Multiplication
of FPIBEL
Virus at 31”
Virus was grown at 31” in order to slow down multiplication. Production of hae-
magglutin (HA) in infected chick embryo (CE) cells at 31” had a latent period of between 2 and 3 hr (Fig. 1). When actinomycin D (AMD) was added at hourly intervals after infection, it was found to inhibit HA production by over 97% when added at 2 hr post infection. Determination of the Major Type of VirusSpecific RNA Present in Cells Infected with FPIBEL at 31” In addition to cRNA, cells infected with influenza virus contain newly synthesized virus-specific RNA which is identical to that found in the virus particle (vRNA) (Nayak and Baluda, 1968; Scholtissek and Rott, 1970). In experiments performed at 37”, it was found that early in infection synthesis of cRNA predominates, but that by 3-6 hr postinfection synthesis of vRNA is greater (Scholtissek and Rott, 1970; Krug, 1972; Avery, 1974). Thus the proportion of intracellular cRNA falls as infection proceeds. In order to determine whether cRNA was still present in excess at 6 hr after infection with FP/BEL at 31”, the procedure de-
FIG. 1. Growth of FP/BEL in CE cells infected at room temperature with 10 PFU/cell. (a) Infected cells, maintained at 31” (O--i>) or 37” (04) were harvested at hourly intervals into the medium, sonicated and assayed for total HA. (b) AMD (1 &ml) was added at hourly intervals from 0 hr to infected cells maintained at 31”. Cells were harvested 24 hr after infection and assayed for HA as in (a). Arrows represent undetectable levels of HA.
CONTROL OF INFLUENZA
scribed by Avery (1974) was employed. Infected cells were labelled with [“Hluridine from zero time and harvested 6 hr postinfection. RNA was extracted from the infected cells and annealed either alone or with unlabelled virion RNA added in excess. The results obtained (Table 1) showed that annealing was increased by addition of excess unlabelled virion RNA. This means that cRNA is present in excess over vRNA even at 6 hr postinfection at 31”. Temporal
Control of Transcription
In the experiment detailed below, 32Plabelled influenza virion RNA was used as a probe to investigate the complementary RNA sequences present in infected cells. RNA was extracted from infected cells at 2 hr and at 6 hr postinfection. Increasing quantities of these RNA’s were annealed with 32P-labelled RNA extracted from influenza virus particles. Figure 2 (open TABLE 1 ANNEALING OF [9H]U~~~~~ LABELLEDRNA EXTRACTED FROMINFECTEDCELLS 6 HR POSTINFECTION
Annealing conditions
% Ribonuclease resistance
Selfannealed Annealed with 40 pg of virion RNA
10.1 32.6
INFECTED
CELL
RNA
lmgl
FIG. 2. Annealing of 9-labelled virion RNA (10’ cpm per sample) with RNA extracted from infected cells at 2 hr (04) or 6 hr (O---O) postinfection. Annealing with equal amounts of 2-hr and 6-hr RNA simultaneously is represented by the triangles.
411
VIRUS TRANSCRIF’TION
circles) shows that an increasing amount of the ‘*P-labelled virion RNA became ribonuclease resistant as more of the RNA extracted at 6 hr postinfection was added. No plateau representing saturation of the vRNA with unlabelled cRNA was obtained, even though RNA from 1.5 x lo9 infected cells was used. However, RNA extracted at 2 hr postinfection showed quite different annealing characteristics. As increasing quantities were hybridized with the labelled virion RNA there was an initial rise in ribonuclease resistance which was followed by a plateau indicating that all the virion RNA capable of forming hybrids had done so. At all concentrations of RNA the annealing obtained with 2-hr RNA was less than that obtained with 6-hr RNA. The fact that more of the 32P-labelled virion RNA can be protected from ribonuclease action by annealing with RNA isolated from cells 6 hr postinfection means that there are more cRNA species present in infected cells at the later time. Figure 2 (triangles) also shows annealing of mixtures of equal amounts of 2- and 6-hr RNA preparations with 32P-labelled virion RNA. In all cases the addition of 2-hr RNA did not significantly increase the annealing found with 6-hr RNA alone. Effect of Actinomycin Transcription
D on FPIBEL
Virus
In order to determine the effect of AMD on the pattern of transcription of influenza virus RNA the following experiments were performed. 1. Chick embryo cells were incubated at 37” with AMD (5 Ng/ml). One hour later virus was added and allowed to adsorb in the presence of the drug at room temperature. The cells were then incubated for 2 hr at 31” before being harvested for RNA extraction. 2. Chick embryo cells were infected and incubated at 31”. After a 2-hr incubation, AMD (5 pg/ml) was added, and the cells were harvested 4 hr later, that is at 6 hr postinfection. RNA was prepared from cells treated with actinomycin D, as described above, for -1 to 2 hr or from 2-4 hr postinfection and annealed with 32P-
412
AVERY
AND DIMMOCK
labelled virion RNA. The results of these experiments are presented in Table 2. The presence of AMD during the first 2 hr of infection has clearly prevented the transcription that occurs in untreated infected cells. In addition, the level of transcription at 6 hr is much reduced compared to that obtained in the untreated infected cultures. Effect of Cycloheximide Transcription
on FPIBEL
Virus
Chick embryo cells were treated with cycloheximide at 100 &ml for 1 hr prior to infection. Infection with virus and subsequent incubation at 31”, both in the presence of the drug, were carried out as described above. Cells were harvested at 2 and 6 hr postinfection and RNA prepared. This RNA was annealed with [“‘PIlabelled virion RNA, and the results are presented in Table 3. The levels of annealing obtained appear to be identical with those found in the absence of cycloheximide, although greater quantities of cycloheximide-treated infected-cell RNA were required to achieve these levels. Essentially the same result was obtained when the experiment was repeated but with cycloheximide present at 300 pglml. These results suggest that cycloheximide has no effect on the temporal pattern of influenza virus transcription. In order to show that cycloheximide was indeed inhibiting proTABLE
2
ANNEALING WITH SZP-L~~~~~~~ INFLUENZA VIRION RNA OF THE UNLABELLED RNA EXTRACTED FROM ACTINOMYCIN D-TREATED CE CELLS AT 2 OR 6 HR POSTINFECTION’
Actinomycin
D-treated
Unlabeled RNA (wz)
% Ribonuclease resistance
Unlabeled RNA (fig)
% Ribonuclease resistance
2hr
6hr
2hr
6hr
2hr
6hr
2hr
6hr
340 425 850
500 625 1,250
1.1
27.6
336
23.4 27.7
839
21.7 26.1 24.2
37.2
1.4 3.6
333 740 1,018
Untreated
610
controP
44.9 49.8
a Actinomycin treatment was from - 1 to 2 hr or 2-6 hr postinfection, as described in the text. * Values taken from Fig. 2.
TABLE
3
ANNEALING WITH 32P-L~~~~~.~~ INFLUENZA VIRION RNA OF THE UNLABELLED RNA EXTRACTED FROM CYCLOHEXIMIDE-TREATED CELLS AT 2 OR 6 HR POSTINFECTION”
Cycloheximide treated (100 pup/ml)
Untreated
control”
Unlabelled RNA bg)
% Ribonuclease resistance
URnr$~el$,d
% Ribonuclease resistance
2hr
2hr
6hr
2hr
2hr
30.7
336 610
6hr
9.9
500 625
11.6
1,000 1,250
23.4
400 500
39.4 46.2
839
6hr 333 740
1,018
a Cycloheximide treatment is detailed b Values taken from Fig. 2.
27.7 26.1
24.2
6hr 37.2
44.9 49.8
in the text.
tein synthesis, the incorporation of radioactively labelled amino acids into proteins in infected CE cells was measured in the presence of varying amounts of the drug. The incorporation obtained between 0 and 1 hr or 0 and 6 hr postinfection is shown in Table 4. Only about 1% of the untreated level of protein synthesis is occurring in the presence of 300 &ml of cycloheximide. To confirm the absence of viral-specified protein synthesis, proteins from cells labelled with [YSlmethionine (Radiochemical Centre, Amersham, England) were analysed by polyacrylamide-gel electrophoresis as described by Kelly et al. (1974). Only small amounts of two proteins were detected, and these were present in both infected cells and noninfected controls. The synthesis of these two proteins was prevented by chloramphenicol, and thus they are presumably mitochondrial in origin. Thus we must conclude that, although cycloheximide completely prevents viruscoded protein synthesis, it does not affect the temporal pattern of viral transcription. DISCUSSION
Our results show that by 2 hr postinfection at 31°, transcription of influenza virus RNA is not complete. Only about 25% of the influenza virus genome can be made ribonuclease resistant by annealing with RNA synthesised in infected cells during
CONTROL TABLE
OF INFLUENZA
4
INCORPORATIONOF +>C-LABELLED AMINO ACIDS INTO INFECTED CE CELLS IN THE PRESENCE OF CYCLOHEXIMIDE Cycl;$;ilmide 20 60 100 200 300
O-l hr
O-6 hr
6.4” 3.8 3.0 2.1 1.4
4.4 3.1 2.2 1.7 1.3
“Data expressed as a percentage of the incorporation in untreated controls.
this time. By 6 hr postinfection more of the genome is being expressed as shown by the increased annealing with RNA prepared at this time. There were insufficient cells available to prepare enough of the latter RNA to saturate and so we cannot say from these data whether the entire genome is transcribed by 6 hr postinfection. However, we have confirmed Skehel’s (1973) observation that all viral proteins normally detectable are synthesised at this time. This would suggest that the complete influenza virus genome is expressed by 6 hr postinfection. As the maximum annealing we observed at this time was about 50%, we estimate the proportion of the genome that is transcribed by 2 hr to be between 25 and 50%. This value is consistent with the fact that the molecular weights of the proteins detected at 2 hr postinfection by Skehel (1973) represent about 40% of the total coding capacity of the virus. Mixtures of RNA extracted at 2 and 6 hr showed no increased annealing compared to the 6-hr sample alone. This indicates that all RNA species transcribed at 2 hr are also transcribed at 6 hr and there is no switch off of any class of messenger RNA. Confirmation of this comes from the fact that the smallest amount of 6-hr RNA anneals to a level higher than the saturation value at 2 hr, suggesting a buildup of early RNA’s in the 6-hr sample. The increased annealing with increasing amounts of 6-hr RNA can then be attributed to RNA not present at 2 hr. The fact that saturation was not achieved at even quite high levels of input 6-hr RNA may imply that
VIRUS TRANSCRIPTION
413
the concentration of the “late” RNA’s is low in infected cells. The presence of AMD during the first 2 hr of infection completely prevented transcription of influenza RNA. A similar effect on primary transcription has been described by Bean and Simpson (1973). This may represent the primary effect of actinomycin D, which is well known to prevent influenza virus multiplication if added early in infection (Barry et al., 1962). The presence of actinomycin D from 2-6 hr postinfection had the effect of preventing the increase in extent of transcription that occurred in its absence. Thus RNA extracted at 6 hr postinfection in the presence of the drug would only saturate about 25% of the influenza genome. This finding is completely consistent with Skehel’s observation that AMD added at 2 hr prevents the detection of any proteins other than those already present at 2 hr. So it appears that AMD in some way prevents the appearance of the “late” messenger RNA’s. The fact that transcripts of viral RNA are present after 4-hr incubation in the presence of AMD suggests that early cRNA synthesis once initiated can continue in its presence or that cRNA once made is stable. It has been shown that cRNA synthesis does occur in the presence of AMD (Avery, 1974), although at a reduced level (Scholtissek and Rott, 1970). However, Pons (1973) has suggested that AMD completely prevents synthesis of influenza cRNA when added at any time postinfection, and Gregoriades (1970) has presented evidence that influenza messenger RNA is very stable. Thus RNA present at 6 hr may have been made in the first 2 hr of infection. Experiments with cycloheximide indicated that it does not prevent an increase in the extent of annealing of RNA prepared at 6 hr compared with that extracted at 2 hr postinfection. Thus it would seem that protein synthesis may not be necessary for the switch from early to early- and-late messenger RNA synthesis. Skehel (1973) reported that on removal of cycloheximide the three early proteins were rapidly labelled while the others showed a delay of 30 min in incorporation of radioactivity. He
414
AVERY AND DIMMOCK
interpreted this to mean that messenger RNA for the early proteins was already present and that messenger RNA coding for the other proteins could not be synthesised in the presence of cycloheximide. The results presented above do not support this conclusion. However, it is apparent (Table 3) that more of the RNA from cycloheximide-treated cells must be added to produce annealing equivalent to that found with RNA from untreated cultures. This presumably reflects an overall decreased rate of RNA synthesis in the presence of cycloheximide (as observed by Bean and Simpson (1973) and Stephenson and Dimmock (1975) as the amplification of the polymerase activity is prevented. RNA species that are synthesised before 2 hr postinfection would be expected to be present in greater amounts than those whose synthesis begins later than this. The delay observed by Skehel may reflect the need for the late RNA’s to reach a level where the incorporation they stimulate is detectable. In conclusion, our results indicate a temporal control of influenza virus transcription that is inhibited by actinomycin D but not by cycloheximide. ACKNOWLEDGMENTS We thank Mrs. J. S. Jones and Mr. M. R. Lee for excellent technical assistance and Professor D. C. Burke for his interest and criticism. REFERENCES AVERY, R. J. (1974). The subcellular localization of virus-specific RNA in influenza virus infected cells. J. Gen. Virol. 24, 77-88. BARRY,R. D., IVES, D. R., and CRUICKSHANK, J. G. (1962). Participation of deoxyribonucleic acids in the multiplication of influenza virus. Nature (London) 194, 1139-1140. BEAN, W. J., JR., and SIMPSON,R. W. (1973). Primary transcription of the influenza virus genome in
permissive cells. Virology 56,646-651. BISHOP, D. H. L., ROY, P., BEAN, W. J., JR., and SIMPSON,R. W. (1972). Transcription of the influenza RNA genome by a virion polymerase. J. Virol. 10, 689-697. CHOW,N., and SIMPSON,R. W. (1971). RNA-dependent RNA polymerase activity associated with virions and sub-viral particles of myxoviruses. Proc. Nat. Acad. Sci. USA 68, 752-756. DUESBERG, P. H. (1968). The RNAs of influenza virus. Proc. Nat. Acad. Sci. USA 59, 930-937. GREGORIADES, A. (1970). Actinomycin D and influenza virus multiplication in the chick embryo fibroblast. Virology 42,905-916. KELLY, D. C., AVERY, R. J., and DIMMOCK, N. J. (1974). Failure of an influenza virus to initiate infection in enucleate BHK cells. J. Virol. 13, 1155-1161. KINGSBURY,D. W., and WEBSTER,R. G. (1973) Cellfree translation of influenza virus messenger RNA. Virology 56, 654-657. MORSER,M. J., KENNEDY,S. I. T., and BURKE,D. C. (1973). Virus specified polypeptides in cells infected with Semliki Forest virus. J. Gen. Virol. 21,19-29. NAYAK, D. P., and BALUDA,M. A. (1968). Ribonucleic acid synthesis in cells infected with influenza virus. J. Virol. 2, 99-109. PONS, M. W. (1972). Studies on the replication of influenza virus RNA. Virology 47, 823-1832. PONS,M. W. (1973). The inhibition of influenza virus RNA synthesis by actinomycin D and cycloheximide. Virology 51, 120-128. PONS,M. W., and HIRST, G. K. (1968). Polyacrylamide gel electrophoresis of influenza virus RNA. Virology 34, 385-388. SCHOLTISSEK, C., and ROTT, R. (1970). Synthesis in uiuo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40, 989496. STEPHENSON, J. R., and DIMMOCK,N. J. (1975). Early events in influenza virus multiplication: location and fate of the input RNA. Virology, in press. SKEHEL, J. J. (1971). Estimations of the molecular weight of the influenza virus genome. J. Gen. Virol. 11, 103-109. SKEHEL,J. J. (1973). Early polypeptide synthesis in influenza virus-infected cells. Virology 56,394-399.
(1975)
Temporal
Control
of Transcription
of Influenza
Virus RNA
ROGER J. AVERY AND NIGEL J. DIMMOCK Department
of Biological
Sciences, University Accepted
of Warwick, Coventry CV4 7AL, England
November
20, 1974
Only part of the single-stranded RNA genome of influenza virus is transcribed early in infection. The appearance of the RNA synthesised later in infection is prevented by actinomycin D but not by cycloheximide. INTRODUCTION
In many cases it has been shown that the expression of the information present in a viral genome occurs in a controlled fashion. Thus the successive and orderly synthesis of distinct classes of RNA is known in both phage and animal virus-infected cells. Influenza virus possesses a segmented single-stranded genome (Duesberg, 1968; Pons and Hirst, 1968; Skehel, 1971) which is transcribed into complementary RNA (cRNA) in infected cells (Bean and Simpson, 1974; Stephenson and Dimmock, 1975). This cRNA has been implicated as influenza virus messenger RNA (Pons, 1972; Kingsbury and Webster, 1973). Virus particles contain a virion polymerase that in vitro makes RNA complementary to the genome (Chow and Simpson, 1971), and this may well be the enzyme responsible for at least some transcription in uiuo. In vitro studies have shown that this enzyme can copy all or nearly all of the viral genome into cRNA (Bishop et al., 1972). Thus influenza virus might be expected to express all its genetic information immediately on infection. However, Skehel (1973) has found that early in infection with influenza virus only three virus-specific proteins are synthesized but at later times all the viral proteins are present. This clearly indicates that influenza virus does exert temporal control over the expression of its genetic information. The important question which then remains to be asked is at what stage does this control occur? If all 409 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
influenza virus messenger RNA’s are made throughout the growth cycle, then control must be exerted on the translation of these RNA’s. On the other hand, transcription may itself be controlled in uiuo. In order to resolve this problem we have examined the cRNA synthesised early in infection by influenza virus and determined what proportion of the viral genome is represented in these transcripts at various times. MATERIALS
AND METHODS
Virus. A recombinant influenza virus strain between A/FPV/Dutch/27 (Hav 1 Neq 1) and AO/BEL/42 (HON 1) designated FPV/BEL was grown in embryonated chicken’s eggs (Stephenson and Dimmock, 1975). Growth of 32P-labelled virus in deem bryonated eggs. The method for prepar-
ing infectious virus with a specific activity of about 80,000 cpm/pg of RNA has been described (Stephenson and Dimmock, 1975). Before extracting the RNA the virus was dialysed against 0.1 x SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). Growth
of virus
in chick embryo cells.
Chick embryo cells, 7.5 x lo7 per 14-cm petri dish (Morser et al., 1973) were infected with virus at an input multiplicity of 10 PFU/cell, and virus and was allowed to adsorb for 45 min at room temperature. The cells were washed with maintenance medium at 31” and then incubated in 15
410
AVERY
AND DIMMOCK
ml of this medium at the same temperature. Maintenance medium consisted of 2% calf serum in Medium 199 buffered with bicarbonate. At 2 or 6 hr postinfection the medium was removed and the cells were washed twice with phosharvested, phate-buffered saline and resuspended in 0.01 M NaCl, 0.001 M EDTA and 0.01 M sodium acetate, pH 5.2. When labelled RNA was required, maintenance medium was replaced by bicarbonate-buffered Earle’s saline containing 2% dialysed calf serum, and 15 PCi of [3H]uridine per ml. Preparation of RNA. RNA was prepared from virus and infected cells as described by Avery (1974). Hybridization. All RNA samples to be hybridized were dissolved in 0.04 x SSC. Radioactively labelled virus-particle RNA and unlabeled RNA extracted from infected cells were mixed and denatured by heating at 98” for 4 min and then rapidly brought to 6 x SSC and 65”. Incubation was carried on for 4 hr at this temperature. Subsequent procedures have been described previously (Avery, 1974)) except that T, ribonuclease was used at a concentration of 850 units/ml. Radioactively labelled virion RNA showed between 0 and 0.5% ribonuclease resistance, and this was not increased on selfannealing. Measurement of protein synthesis in the presence of cycloheximide. Chick embryo cells (9 x lo6 cells per 5-cm dish) were pretreated for 1 hr with various amounts of cycloheximide at 37”. Virus was adsorbed in the presence of the drug at room temperature. Infected cells were placed at 31’ and 2.5 PCi of a ‘“C-labelled amino acid mixture (57 mCi/mAtom carbon, The Radiochemical Centre, Amersham) was added per plate (zero time), Cells were precipitated with ice-cold 5% TCA at 1 or 6 hr postinfection, collected on cellulose acetate filters, and washed three times with 5% TCA and once with ether. Filters were dried and counted. RESULTS
Multiplication
of FPIBEL
Virus at 31”
Virus was grown at 31” in order to slow down multiplication. Production of hae-
magglutin (HA) in infected chick embryo (CE) cells at 31” had a latent period of between 2 and 3 hr (Fig. 1). When actinomycin D (AMD) was added at hourly intervals after infection, it was found to inhibit HA production by over 97% when added at 2 hr post infection. Determination of the Major Type of VirusSpecific RNA Present in Cells Infected with FPIBEL at 31” In addition to cRNA, cells infected with influenza virus contain newly synthesized virus-specific RNA which is identical to that found in the virus particle (vRNA) (Nayak and Baluda, 1968; Scholtissek and Rott, 1970). In experiments performed at 37”, it was found that early in infection synthesis of cRNA predominates, but that by 3-6 hr postinfection synthesis of vRNA is greater (Scholtissek and Rott, 1970; Krug, 1972; Avery, 1974). Thus the proportion of intracellular cRNA falls as infection proceeds. In order to determine whether cRNA was still present in excess at 6 hr after infection with FP/BEL at 31”, the procedure de-
FIG. 1. Growth of FP/BEL in CE cells infected at room temperature with 10 PFU/cell. (a) Infected cells, maintained at 31” (O--i>) or 37” (04) were harvested at hourly intervals into the medium, sonicated and assayed for total HA. (b) AMD (1 &ml) was added at hourly intervals from 0 hr to infected cells maintained at 31”. Cells were harvested 24 hr after infection and assayed for HA as in (a). Arrows represent undetectable levels of HA.
CONTROL OF INFLUENZA
scribed by Avery (1974) was employed. Infected cells were labelled with [“Hluridine from zero time and harvested 6 hr postinfection. RNA was extracted from the infected cells and annealed either alone or with unlabelled virion RNA added in excess. The results obtained (Table 1) showed that annealing was increased by addition of excess unlabelled virion RNA. This means that cRNA is present in excess over vRNA even at 6 hr postinfection at 31”. Temporal
Control of Transcription
In the experiment detailed below, 32Plabelled influenza virion RNA was used as a probe to investigate the complementary RNA sequences present in infected cells. RNA was extracted from infected cells at 2 hr and at 6 hr postinfection. Increasing quantities of these RNA’s were annealed with 32P-labelled RNA extracted from influenza virus particles. Figure 2 (open TABLE 1 ANNEALING OF [9H]U~~~~~ LABELLEDRNA EXTRACTED FROMINFECTEDCELLS 6 HR POSTINFECTION
Annealing conditions
% Ribonuclease resistance
Selfannealed Annealed with 40 pg of virion RNA
10.1 32.6
INFECTED
CELL
RNA
lmgl
FIG. 2. Annealing of 9-labelled virion RNA (10’ cpm per sample) with RNA extracted from infected cells at 2 hr (04) or 6 hr (O---O) postinfection. Annealing with equal amounts of 2-hr and 6-hr RNA simultaneously is represented by the triangles.
411
VIRUS TRANSCRIF’TION
circles) shows that an increasing amount of the ‘*P-labelled virion RNA became ribonuclease resistant as more of the RNA extracted at 6 hr postinfection was added. No plateau representing saturation of the vRNA with unlabelled cRNA was obtained, even though RNA from 1.5 x lo9 infected cells was used. However, RNA extracted at 2 hr postinfection showed quite different annealing characteristics. As increasing quantities were hybridized with the labelled virion RNA there was an initial rise in ribonuclease resistance which was followed by a plateau indicating that all the virion RNA capable of forming hybrids had done so. At all concentrations of RNA the annealing obtained with 2-hr RNA was less than that obtained with 6-hr RNA. The fact that more of the 32P-labelled virion RNA can be protected from ribonuclease action by annealing with RNA isolated from cells 6 hr postinfection means that there are more cRNA species present in infected cells at the later time. Figure 2 (triangles) also shows annealing of mixtures of equal amounts of 2- and 6-hr RNA preparations with 32P-labelled virion RNA. In all cases the addition of 2-hr RNA did not significantly increase the annealing found with 6-hr RNA alone. Effect of Actinomycin Transcription
D on FPIBEL
Virus
In order to determine the effect of AMD on the pattern of transcription of influenza virus RNA the following experiments were performed. 1. Chick embryo cells were incubated at 37” with AMD (5 Ng/ml). One hour later virus was added and allowed to adsorb in the presence of the drug at room temperature. The cells were then incubated for 2 hr at 31” before being harvested for RNA extraction. 2. Chick embryo cells were infected and incubated at 31”. After a 2-hr incubation, AMD (5 pg/ml) was added, and the cells were harvested 4 hr later, that is at 6 hr postinfection. RNA was prepared from cells treated with actinomycin D, as described above, for -1 to 2 hr or from 2-4 hr postinfection and annealed with 32P-
412
AVERY
AND DIMMOCK
labelled virion RNA. The results of these experiments are presented in Table 2. The presence of AMD during the first 2 hr of infection has clearly prevented the transcription that occurs in untreated infected cells. In addition, the level of transcription at 6 hr is much reduced compared to that obtained in the untreated infected cultures. Effect of Cycloheximide Transcription
on FPIBEL
Virus
Chick embryo cells were treated with cycloheximide at 100 &ml for 1 hr prior to infection. Infection with virus and subsequent incubation at 31”, both in the presence of the drug, were carried out as described above. Cells were harvested at 2 and 6 hr postinfection and RNA prepared. This RNA was annealed with [“‘PIlabelled virion RNA, and the results are presented in Table 3. The levels of annealing obtained appear to be identical with those found in the absence of cycloheximide, although greater quantities of cycloheximide-treated infected-cell RNA were required to achieve these levels. Essentially the same result was obtained when the experiment was repeated but with cycloheximide present at 300 pglml. These results suggest that cycloheximide has no effect on the temporal pattern of influenza virus transcription. In order to show that cycloheximide was indeed inhibiting proTABLE
2
ANNEALING WITH SZP-L~~~~~~~ INFLUENZA VIRION RNA OF THE UNLABELLED RNA EXTRACTED FROM ACTINOMYCIN D-TREATED CE CELLS AT 2 OR 6 HR POSTINFECTION’
Actinomycin
D-treated
Unlabeled RNA (wz)
% Ribonuclease resistance
Unlabeled RNA (fig)
% Ribonuclease resistance
2hr
6hr
2hr
6hr
2hr
6hr
2hr
6hr
340 425 850
500 625 1,250
1.1
27.6
336
23.4 27.7
839
21.7 26.1 24.2
37.2
1.4 3.6
333 740 1,018
Untreated
610
controP
44.9 49.8
a Actinomycin treatment was from - 1 to 2 hr or 2-6 hr postinfection, as described in the text. * Values taken from Fig. 2.
TABLE
3
ANNEALING WITH 32P-L~~~~~.~~ INFLUENZA VIRION RNA OF THE UNLABELLED RNA EXTRACTED FROM CYCLOHEXIMIDE-TREATED CELLS AT 2 OR 6 HR POSTINFECTION”
Cycloheximide treated (100 pup/ml)
Untreated
control”
Unlabelled RNA bg)
% Ribonuclease resistance
URnr$~el$,d
% Ribonuclease resistance
2hr
2hr
6hr
2hr
2hr
30.7
336 610
6hr
9.9
500 625
11.6
1,000 1,250
23.4
400 500
39.4 46.2
839
6hr 333 740
1,018
a Cycloheximide treatment is detailed b Values taken from Fig. 2.
27.7 26.1
24.2
6hr 37.2
44.9 49.8
in the text.
tein synthesis, the incorporation of radioactively labelled amino acids into proteins in infected CE cells was measured in the presence of varying amounts of the drug. The incorporation obtained between 0 and 1 hr or 0 and 6 hr postinfection is shown in Table 4. Only about 1% of the untreated level of protein synthesis is occurring in the presence of 300 &ml of cycloheximide. To confirm the absence of viral-specified protein synthesis, proteins from cells labelled with [YSlmethionine (Radiochemical Centre, Amersham, England) were analysed by polyacrylamide-gel electrophoresis as described by Kelly et al. (1974). Only small amounts of two proteins were detected, and these were present in both infected cells and noninfected controls. The synthesis of these two proteins was prevented by chloramphenicol, and thus they are presumably mitochondrial in origin. Thus we must conclude that, although cycloheximide completely prevents viruscoded protein synthesis, it does not affect the temporal pattern of viral transcription. DISCUSSION
Our results show that by 2 hr postinfection at 31°, transcription of influenza virus RNA is not complete. Only about 25% of the influenza virus genome can be made ribonuclease resistant by annealing with RNA synthesised in infected cells during
CONTROL TABLE
OF INFLUENZA
4
INCORPORATIONOF +>C-LABELLED AMINO ACIDS INTO INFECTED CE CELLS IN THE PRESENCE OF CYCLOHEXIMIDE Cycl;$;ilmide 20 60 100 200 300
O-l hr
O-6 hr
6.4” 3.8 3.0 2.1 1.4
4.4 3.1 2.2 1.7 1.3
“Data expressed as a percentage of the incorporation in untreated controls.
this time. By 6 hr postinfection more of the genome is being expressed as shown by the increased annealing with RNA prepared at this time. There were insufficient cells available to prepare enough of the latter RNA to saturate and so we cannot say from these data whether the entire genome is transcribed by 6 hr postinfection. However, we have confirmed Skehel’s (1973) observation that all viral proteins normally detectable are synthesised at this time. This would suggest that the complete influenza virus genome is expressed by 6 hr postinfection. As the maximum annealing we observed at this time was about 50%, we estimate the proportion of the genome that is transcribed by 2 hr to be between 25 and 50%. This value is consistent with the fact that the molecular weights of the proteins detected at 2 hr postinfection by Skehel (1973) represent about 40% of the total coding capacity of the virus. Mixtures of RNA extracted at 2 and 6 hr showed no increased annealing compared to the 6-hr sample alone. This indicates that all RNA species transcribed at 2 hr are also transcribed at 6 hr and there is no switch off of any class of messenger RNA. Confirmation of this comes from the fact that the smallest amount of 6-hr RNA anneals to a level higher than the saturation value at 2 hr, suggesting a buildup of early RNA’s in the 6-hr sample. The increased annealing with increasing amounts of 6-hr RNA can then be attributed to RNA not present at 2 hr. The fact that saturation was not achieved at even quite high levels of input 6-hr RNA may imply that
VIRUS TRANSCRIPTION
413
the concentration of the “late” RNA’s is low in infected cells. The presence of AMD during the first 2 hr of infection completely prevented transcription of influenza RNA. A similar effect on primary transcription has been described by Bean and Simpson (1973). This may represent the primary effect of actinomycin D, which is well known to prevent influenza virus multiplication if added early in infection (Barry et al., 1962). The presence of actinomycin D from 2-6 hr postinfection had the effect of preventing the increase in extent of transcription that occurred in its absence. Thus RNA extracted at 6 hr postinfection in the presence of the drug would only saturate about 25% of the influenza genome. This finding is completely consistent with Skehel’s observation that AMD added at 2 hr prevents the detection of any proteins other than those already present at 2 hr. So it appears that AMD in some way prevents the appearance of the “late” messenger RNA’s. The fact that transcripts of viral RNA are present after 4-hr incubation in the presence of AMD suggests that early cRNA synthesis once initiated can continue in its presence or that cRNA once made is stable. It has been shown that cRNA synthesis does occur in the presence of AMD (Avery, 1974), although at a reduced level (Scholtissek and Rott, 1970). However, Pons (1973) has suggested that AMD completely prevents synthesis of influenza cRNA when added at any time postinfection, and Gregoriades (1970) has presented evidence that influenza messenger RNA is very stable. Thus RNA present at 6 hr may have been made in the first 2 hr of infection. Experiments with cycloheximide indicated that it does not prevent an increase in the extent of annealing of RNA prepared at 6 hr compared with that extracted at 2 hr postinfection. Thus it would seem that protein synthesis may not be necessary for the switch from early to early- and-late messenger RNA synthesis. Skehel (1973) reported that on removal of cycloheximide the three early proteins were rapidly labelled while the others showed a delay of 30 min in incorporation of radioactivity. He
414
AVERY AND DIMMOCK
interpreted this to mean that messenger RNA for the early proteins was already present and that messenger RNA coding for the other proteins could not be synthesised in the presence of cycloheximide. The results presented above do not support this conclusion. However, it is apparent (Table 3) that more of the RNA from cycloheximide-treated cells must be added to produce annealing equivalent to that found with RNA from untreated cultures. This presumably reflects an overall decreased rate of RNA synthesis in the presence of cycloheximide (as observed by Bean and Simpson (1973) and Stephenson and Dimmock (1975) as the amplification of the polymerase activity is prevented. RNA species that are synthesised before 2 hr postinfection would be expected to be present in greater amounts than those whose synthesis begins later than this. The delay observed by Skehel may reflect the need for the late RNA’s to reach a level where the incorporation they stimulate is detectable. In conclusion, our results indicate a temporal control of influenza virus transcription that is inhibited by actinomycin D but not by cycloheximide. ACKNOWLEDGMENTS We thank Mrs. J. S. Jones and Mr. M. R. Lee for excellent technical assistance and Professor D. C. Burke for his interest and criticism. REFERENCES AVERY, R. J. (1974). The subcellular localization of virus-specific RNA in influenza virus infected cells. J. Gen. Virol. 24, 77-88. BARRY,R. D., IVES, D. R., and CRUICKSHANK, J. G. (1962). Participation of deoxyribonucleic acids in the multiplication of influenza virus. Nature (London) 194, 1139-1140. BEAN, W. J., JR., and SIMPSON,R. W. (1973). Primary transcription of the influenza virus genome in
permissive cells. Virology 56,646-651. BISHOP, D. H. L., ROY, P., BEAN, W. J., JR., and SIMPSON,R. W. (1972). Transcription of the influenza RNA genome by a virion polymerase. J. Virol. 10, 689-697. CHOW,N., and SIMPSON,R. W. (1971). RNA-dependent RNA polymerase activity associated with virions and sub-viral particles of myxoviruses. Proc. Nat. Acad. Sci. USA 68, 752-756. DUESBERG, P. H. (1968). The RNAs of influenza virus. Proc. Nat. Acad. Sci. USA 59, 930-937. GREGORIADES, A. (1970). Actinomycin D and influenza virus multiplication in the chick embryo fibroblast. Virology 42,905-916. KELLY, D. C., AVERY, R. J., and DIMMOCK, N. J. (1974). Failure of an influenza virus to initiate infection in enucleate BHK cells. J. Virol. 13, 1155-1161. KINGSBURY,D. W., and WEBSTER,R. G. (1973) Cellfree translation of influenza virus messenger RNA. Virology 56, 654-657. MORSER,M. J., KENNEDY,S. I. T., and BURKE,D. C. (1973). Virus specified polypeptides in cells infected with Semliki Forest virus. J. Gen. Virol. 21,19-29. NAYAK, D. P., and BALUDA,M. A. (1968). Ribonucleic acid synthesis in cells infected with influenza virus. J. Virol. 2, 99-109. PONS, M. W. (1972). Studies on the replication of influenza virus RNA. Virology 47, 823-1832. PONS,M. W. (1973). The inhibition of influenza virus RNA synthesis by actinomycin D and cycloheximide. Virology 51, 120-128. PONS,M. W., and HIRST, G. K. (1968). Polyacrylamide gel electrophoresis of influenza virus RNA. Virology 34, 385-388. SCHOLTISSEK, C., and ROTT, R. (1970). Synthesis in uiuo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40, 989496. STEPHENSON, J. R., and DIMMOCK,N. J. (1975). Early events in influenza virus multiplication: location and fate of the input RNA. Virology, in press. SKEHEL, J. J. (1971). Estimations of the molecular weight of the influenza virus genome. J. Gen. Virol. 11, 103-109. SKEHEL,J. J. (1973). Early polypeptide synthesis in influenza virus-infected cells. Virology 56,394-399.
