Transcription and replication of influenza virus RNA

VIROLOGY 98, 211-225 (1979)

Transcription and Replication of Influenza Virus RNA

THOMAS BARRETT, ADRIAN J. WOLSTENHOLME, AND BRIAN W. J. MAHY’

Division of Virology, Department of Pathology, University of Cambridge, Laboratories Block,

Addenhoke’s Hospital, Hills Road, Cambridge CB2 2QQ, England

Accepted May 22, 1979

Methods are described for measuring the amounts of virus-specific polyadenylated (A+) cRNA, nonpolyadenylated (A-) cRNA, and virus genome RNA (vRNA) in nucleus and cytoplasm of cells infected with influenza virus. The amounts of A(+) cRNA to individual virus genes accumulated during infection at different rates, not related to gene molecular weight, indicating transcriptional control. The pattern of accumulation of each gene transcript was the same in the nucleus as in the cytoplasm. Addition of cycloheximide during infection abolished the transcriptional control and resulted in linear accumulation of each gene transcript. In cells treated with 0.1 pg/ml actinomycin D, which blocked late but not early virus-specific protein synthesis, there was a significant accumulation of all gene transcripts in the nucleus and a relative decrease in transcripts of genes coding for late proteins (M and HA) in the cytoplasm. Analysis of A(-) cRNA and vRNA accumulations in infected cells showed that the A(-) cRNA synthesis precedes vRNA synthesis and net accumulation of A(-) cRNA ceased by 3 hr postinfection (pi). Significant amounts of vRNA in excess of input vRNA were detected by about 2 hr pi and continued to accumulate in both nucleus and cytoplasm at least up to 6 hr pi. Protein synthesis was required for A(-) cRNA production, since in the presence of cycloheximide very little A(-) cRNA, and no vRNA, could be detected. Actinomycin D (0.1 pg/ml) reduced the amount of A(-) cRNA, and completely inhibited the increase in vRNA which occurred from 2 hr pi in normal infection.

INTRODUCTION

The influenza virus genome consists of eight single-stranded RNA segments of negative polarity with respect to functional mRNA (for review see Barry and Mahy, 1979). Early after infection these RNA segments (vRNA)~ are transcribed by a virion- associated RNA transcriptase into two classes of complementary RNA (cRNA), a polyadenylated form which associates with cell polysomes and serves as mRNA, and a

1 To whom reprint requests should be addressed. * Abbreviations used: CEF, chick embryo fibro-

blasts; FPV, fowl plague virus; HA, haemagglutinin; M, matrix protein; NA, neuraminidase; NP, nucleo- protein; NS, nonstructural protein; PBS, phosphate- buffered saline; RSB, buffer containing 10 mM NaCl, 1.5 mM MgC&, 10 m&f Tris-HCl, pH 7.4, SDS, sodium dodecyl sulfate; TE, buffer containing, 1 mM EDTA and 10 mM Tris-HCl, pH 7.4; vRNA, virus genome RNA; A(+) cRNA, polyadenylated RNA complementary to vRNA; A(-) cRNA, nonpolyadenylated RNA complementary to vRNA; pi, post infection.

nonpolyadenylated form which may be the template for new vRNA synthesis (Hay et al., 1977). Recent evidence suggests that transcription is regulated with respect to the amounts of each functional mRNA present in the cytoplasm at different times after infection (Hay et al., 197’7; Inglis and Mahy, 1979). The mechanism controlling transcription is not understood, but since there is considerable evidence that a functional cell nucleus is required for this proc- ess (Follett et al., 1974; Kelly et al., 1974) and that the nucleus may be the site at which transcription occurs (Armstrong and Barry, 1974; Barrett et al., 1978; Taylor et al., 1977), we have investigated the possibility that differences exist in the relative amounts of each polyadenylated gene transcript at various times postinfection and between the nucleus and cytoplasm. We have studied the accumulation of nonpolyadenylated cRNA and of vRNA in each cell fraction at different times following virus infec-

211 0042~6322/79/130211-15$02.00/0 Copyright Q 19’79 by Academic Press, Inc. All rights of reproduction in any form reserved.

212 BARRETT, WOLSTENHOLME, AND MAHY

tion. The effects of cycloheximide and actinomycin D on the different classes of virus-specific RNA were also investigated.

MATERIALS AND METHODS

Cells and virus. Monolayer cultures of CEF cells, grown in plastic petri dishes, were prepared as previously described (Borland and Mahy, 1968). Stocks of influenza A virus (FPV, Restock strain) were grown in 11-day fertile hen eggs; the eggs were infected with approximately lo3 PFU of virus and the allantoic fluid was harvested after 24 hr of growth when the virus titer was greater than lo9 PFU/ml. For infection, cells were washed in phosphate-buffered saline (PBS), then overlaid with this allantoic fluid diluted 1 in 4 with PBS (approximate m.o.i. 50 PFU/cell). After 30 min of adsorption at room temperature, the cells were drained, overlaid with medium 199 containing 2% calf serum, and incubated at 37” (zero time).

DmLg treatments. Confluent CEF cells were pretreated for 1 hr with medium containing the appropriate concentration of either actinomycin D or cycloheximide. In all cases 0.1 pg/ml actinomycin D was used. This is not sufficient to completely inhibit virus transcription but appears to affect pri- marily host nucleolar function (Rickinson and Dendy, 1969). Cycloheximide was used at 100 pg/ml which is sufficient to inhibit cell protein synthesis by at least 98% (Lamb and Choppin, 1976). Virus adsorption was carried out in the presence of the same concentration of drug added to the virus sus- pension. After 30 min of adsorption at room temperature, the cell monolayers were drained, overlaid with medium 199 containing 2% calf serum and the appropriate amount of drug, and incubated at 37”.

Cell fractionation. At various times postinfection, the medium was removed from the cells and replaced with ice-cold PBS. The cells were harvested by scraping with a rubber policeman and pelleted by centrifugation at 1000 g for 2 min. The cell pellets were resuspended in B4 buffer (10 mM NaCl, 1.6 mM MgCl%, 1 mM triethanol- amine, 10 mM Tris-HCl, pH 7.4), kept at 0” for 15 min, then homogenized by 15

strokes of a Dounce homogenizer. Crude nuclei were then separated by centrifugation at 1000 g for 2 min. The cytoplasmic supernatant was kept at 0” while the nuclear pellet was resuspended in RSB (10 mM NaCl, 1.5 mM MgC&, 10 mM Tris-HCI, pH 7.4) containing sodium deoxycholate (0.2% w/v) and Nonidet P-40 (1% v/v). After thorough mixing for 1 min the nuclei were again separated by centrifugation at 1000 g for 2 min. The supernatant was combined with the previous cytoplasmic supernatant and this fraction was used as the cytoplasmic fraction.

The nuclear pellet was washed three times by resuspension and centrifugation in 0.32 M sucrose, 1 mM MgCl*, and the final nuclear pellet was resuspended in RSB.

Extraction of RNA. The nuclear and cytoplasmic fractions were each mixed with equal volumes of 1 mg/ml Pronase in buffer (0.05 M NaCI, 0.01 M EDTA, 0.5% SDS, 0.1 M Tris-HCI, pH 7.5) and incubated for 60 min at 37”. The aqueous phase was twice extracted at 37” with equal volumes of chloroform-isoamylalcohol (25:l) and the final aqueous phase precipitated overnight, after addition of 2.5 vol ethanol, at -20”. The precipitated nucleic acid fractions from the nuclei were collected by centrifugation and resuspended in 0.1 M NaCl, 0.015 M MgC&, 0.1 M Tris-HCI, pH 7.4, and incubated with DNase I (50 kg/ml) for 2 hr at 28”. RNA was then purified from the mixture by Pronase treatment, chloroform- isoamylalcohol extraction, and precipitation in ethanol at -20” as before. In some cases the chloroform-isoamylalcohol extraction was replaced by phenol-chloroform (1:l) extraction. To avoid trapping polyadenylated RNA in the interphase the phenol layer was reextracted with 0.1 M Tris- HCl, pH 9.0, 150 mM NaCl. The RNA pre- cipitates were then collected by centrifugation, dissolved in TE buffer (1 mM EDTA, 10 mM Tris-HCI, pH 7.4), and dimethyl sulfoxide was added to a final concentration of 90% before heating to 45” for 20 min to denature the RNA. The RNA was reprecipitated in ethanol as before. Polyade- nylated and nonpolyadenylated fractions of the RNA were prepared by oligo(dT)-cellulose chromatography.

Separation of polyadenylated RNA. Poly-

INFLUENZA VIRUS RNA SYNTHESIS 21X

adenylated RNA was separated from nonpolyadenylated RNA as described by Glass et al. (1975).

Preparation of injluenxa virus cDNA. Single-strand DNA complementary to purified vRNA (cDNA) was prepared using reverse transcriptase from avian myeloblastosis virus. The reaction mixture contained in a final volume of 100 ~1, 0.1 M Tris-HCl, pH 8.1, 5 mM MgC&, 2 mM each dATP, dCTP, dGTP, 100 $Zi [3H]dTTP (50 Ci/ mmol), 5 mM dithiothreitol, 50 pg/ml actinomycin D, 40-60 pg/ml purified influenza virion RNA, 120 pg/ml oligo(dG) as primer, and 60 units/ml purified reverse transcriptase. The mixture was incubated at 3’7” for 3 hr and then extracted with phenol-chloroform (1:l). Fifty micrograms of yeast tRNA was added to the aqueous phase, which was precipitated by the addition of 2.5 vol ice- cold ethanol. The precipitate was taken up in 0.3 M sodium hydroxide and incubated at 37” for 2 hr to degrade the RNA. Fifty micrograms of yeast tRNA was added, and the mixture neutralised and reprecipitated with ethanol. The final precipitate was taken up in 0.5 ml distilled water.

Preparation of I 125-labeled vRNA. Virus RNA was iodinated using a slight modifica- tion of the method described by Commer- ford (1971). Ten micrograms of RNA was incubated for 10 min at 60” in 50 ~1 of buffer containing 0.04 mM KI, 0.1 M sodium acetate, pH 4.8, 10 mM thallic chloride, 1 mCi KI’*“. The reaction was stopped by the addition of 2 ~1 P-mercaptoethanol. Unstable iodinated uridine residues were deiodinated by incubating the mixture with 200 11 0.2 M ammonium acetate, pH 8.9, containing 0.5% SDS at 50” for 5 min. Carrier tRNA was then added and the RNA precipitated with 2.5 vol ethanol at -20”. The RNA was reprecipitated several times before purification by gel electrophoresis. Specific activities of approximately 10’ cpm/ pg vRNA were obtained.

Gel electrophoresis. Originally electrophoresis was carried out in 2.2% acrylamide-agarose gels as described by McGeoch et al. (1976). Later 2.5% acrylamide-7 M urea gels run in the Tris-borate-EDTA buffer system (TBE) described by Brown- lee and Cartwright (1977) were used, since

they give better resolution of the influenza virus RNAs.

Extraction of RNA from gels. RNA bands were visualized by autoradiography, excised, and the RNA was extracted by homogenization in phenol, essentially as described by Jeppesen et al. (1972).

RNA-RNA annealing procedure. The RNA-RNA annealing procedure was carried out as described by Glass et al. (1975).

DNA-RNA annealing procedure. RNA samples were dissolved in water at a concentration of 2 (cytoplasmic RNAs) or 0.2 mglml (nuclear RNAs) and mixed with about 40,000 cpm of [3H]cDNA. The mixture was heated to 100” for 3 min and cooled to 70”. A prewarmed salt solution was added to bring the mixture to 0.6 M NaCl, 0.04 M Tris (pH 7.4), 2 mM EDTA. Samples were incubated at 70” in sealed plastic reaction vessels and samples removed at in- tervals up to 3000 min after the addition of the salt solution. The samples were chilled at -20”. The extent of hybridization was determined using S, nuclease. Samples were thawed, diluted with buffer containing 0.03 M sodium acetate, pH 4.5, 2 mM zinc sulfate, 0.3 M sodium chloride, 50 pg/ml de- natured carrier DNA, and split into four equal fractions. To two of these fractions was added 20 ~1 S, nuclease solution and all the fractions were incubated at 37” for 30 min. The acid-precipitable radioactivity in each fraction was determined as described by Glass et al. (1975).

Determination of concentration of virus- speci$c A( +) cRNA in RNA extracted from infected cells. The amount of virus-specific A(+) cRNA was determined as described by Mahy et al. (1977). First, for each sample, a range of concentrations of the cell RNA was annealed to a constant amount (about 1000 cpm) of radiolabeled vRNA (see RNA-RNA annealing procedure). From these hybridization analyses (“screening-in curves”) the amount of nuclear or cytoplasmic RNA giving about 25% annealing with the vRNA was selected for use in the subsequent annealing reactions. This amount of the RNA sample was then hybridized with increasing amounts of 1251-labeled vRNA. The amount of virion RNA annealed at saturation by the known amount of cellu-


lar RNA was determined from the intercept on the ordinate of a double-reciprocal plot of this data (see Fig. 1).

Determination of concentration of virus- specije A(-) cRNA and VRNA in RNA extracted from infected cells. In order to measure the amounts of A(-) cRNA and vRNA in infected cells, nonpolyadenylated RNA extracted from these cells was annealed to both 3H-labeled complementary DNA and to lz51-labeled virion RNA. Since there is an appreciable amount of both virus-specific A( -) cRNA and vRNA in these samples one of these reactions will reach equilibrium at a value of 100% nuclease resistance, whereas the other reaction will reach equilibrium at a lower value, 2, which is the ratio of the concentrations of the classes of virus-specific RNA. The annealing reactions were then repeated, but with the inclusion of a known quantity, z, of unlabeled pure vRNA. This altered the position of equilibrium of the reaction to a different value, y. The amount of labeled nucleic acid is insignificant.

Therefore, if V -=x c

and v+x -= Y

c then

x c=- y-x pg

and v = xc pg,

where

v = amount of virus genome RNA,

c = amount of co.mpIementary RNA.

Since the approximate RNA content of the cell fraction is known the number of gene copies can be calculated from the formula:

AR gene copies = - ,

10-9 where

A = amount of virus-specific RNA (fig) in the hybridization mixture,

R = RNA content of the cell fraction (pg); in these experiments we cal-

culated that each nuclear fraction contained 1.5 pg RNA and each cytoplasmic fraction contained 3 pg RNA,

B = total amount of RNA (pg) in the hybridization mixture,

lo-” = the weight of the influenza virus genome (f.&

Examples of this calculation, using actual experimental data, are presented in Fig. 1.

Chemicals and buglers. Actinomyein D was a gift from Merck, Sharp & Dohme Ltd., Hoddesdon, Herts. Cycloheximide, @- mercaptoethanol, sodium dodecyl sulfate (SDS), yeast tRNA, ribonuclease A, and deoxyribonuclease I(RNase-free) were obtained from Sigma Chemical Company, London. Ribonuclease T, (Worthington) was obtained from Cambrian Chemicals, Croydon. Oligo(dT)-cellulose was obtained from Searle Diagnostic, High Wycombe. 1251, carrier free, and [methyZ-3H]thymi- dine-5’-triphosphate (40-60 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, Bucks. S, nuclease (prepared from takadiastase) was a gift from Dr. R. P. Eglin. Reverse transcriptase prepared from avian myeloblastosis virus was a gift from the Office of Program Resources and Logis- tics, Virology Cancer Program, Viral On- cology, Division of Cancer Cause and Pre- vention, National Cancer Institute, Be- thesda, Maryland.

RESULTS

Characteristics of Radioactively Labeled Inflzcenxa VRNA and cDNA

Influenza vRNA labeled with 125I was used to measure the virus-specific cRNA content of polyadenylated RNA extracted from infected cells. The vRNA was separated into its component segments by polyacrylamide gel electrophoresis and the individual segments were then used to measure the concentration of their corresponding A(+) cRNAs (Fig. 2). The segments were separately extracted from the gel and used individually except that segments l-3, coding for the three P proteins, were not suf- ficiently resolved and were extracted as one band. When the isolated segment RNAs

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FIG. 2. Polyacrylamide gel electrophoresis of 9- labeled vRNA. The influenza virus genes are labeled l-8. Electrophoresis was carried out for (A) 23 hr and (B) 5 hr at 250 V in the TBE system described under Materials and Methods.

were reelectrophoresed on polyacrylamide gels the individual bands ran in the expected order of mobility and there was no evidence of cross-contamination, although some degradation attributable to the gel extraction was seen. This degradation would not affect the annealing characteristics. The purity of individual segments obtained by this method was confirmed by direct RNA se- quence analysis (Robertson, 1979). The in- trinsic ribonuclease resistance of the various RNA species varied from 0.8 to 2%.

The [3H]cDNA copy of influenza vRNA was prepared using reverse transcriptase

and an oligo(dG) primer. The cDNA annealed completely to its template in RNA excess hybridization. In DNA excess, the cDNA could protect lz51-vRNA from diges- tion by ribonuclease almost completely, indicating that all the sequences of vRNA were represented in the cDNA (Fig. 3).

No annealing of either radiolabeled influenza vRNA or influenza cDNA could be detected during exhaustive hybridization with RNA extracted from uninfected cells.

Proportion of Virus-Specify A(-+-) cRNA in Nucleus and Cytoplasm

Our previous results (Barrett et al., 1978) showed that at early times the concentration of influenza virus-specific A(+) cRNA sequences was higher in nuclear RNA than in cytoplasmic RNA. The total numbers of copies in the nucleus increased with time up to 2 hr postinfection (pi) and then declined. The number of A(+) cRNA copies in the cytoplasm increased steadily from 0.5 to 2.5 hr pi, and between 2.5 and 3 hr pi there was a dramatic increase in A( +> cRNA concentration. Only at 0.5 hr pi did the absolute number of copies in the nucleus exceed that in the cytoplasm.

The representation of individual copies of vRNA genes 4 to 8 relative to the number of copies of genes l-3 was determined by hybridization of nuclear and cytoplasmic polyadenylated RNA to 1251-labeled vRNA segments. For each time studied, the concentration of RNA hybridizing to band 1 (segments l-3) was given a molar value

.

z “-

L-I 16’ IO+ 10-I IO0

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FIG. 3. Kinetics of hybridization of 9-labeled vRNA to an excess of unlabeled virus cDNA. Hybrid- ization was carried out as described under Materials and Methods.

INFLUENZA VIRUS RNA SYNTHESIS 217

TABLE 1

PROPORTIONS OF INFLUENZA VIRUS A(+) cRNA TRANSCRIPTS IN NUCLEUS AND CYTOPLASM OF INFECTED CELLS AT VARIOUS TIMES POSTINFECTION

Gene No. 1.0

Cytoplasm (hr postinfection)

1.5 2.5 4.5

Nucleus (hr postinfection)

1.5 2.5 4.5 Expected ratio”

1-3” 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

4 1.5 3.5 6.1 5.2 1.8 2.9 7.6 1.3 5 4.3 8.7 18.8 9.5 2.8 8.9 7.5 1.6 6 2.7 7.1 7.0 9.5 2.4 7.9 8.3 1.9

7 7.4 12.9 15.9 24.3 5.2 17.8 25.5 3.7 8 9.9 27.3 30.5 27.7 10.8 32.6 26.5 3.9

a Expected molar ratio if the genes are transcribed in proportion to their molecular weight. ’ Molar ratio of copies of each gene is expressed relative to the P genes which are given a value of 1.0.

of 1.0, and the molar amounts of the other segments was expressed relative to band 1 (Table 1). The amount of each A(+) cRNA produced in infected cells did not simply reflect the size of the gene from which it was transcribed. Early in infection, the NS and NP gene A(+) cRNAs were produced in relatively greater amounts; both reached a peak accumulation at 2.5 hr pi then declined. NA and HA gene A( +) cRNA accumulated slowly over the period studied, while A(+) cRNA complementary to M rose steadily and by 4.5 hr pi was as abundant as NS A( +> cRNA. The accumulation of individual A( +> cRNA segments was similar in the nucleus and the cytoplasm, and there was no evidence of particular segments accumulat- ing in one cell fraction.

Effect of Actinomycin D on A( +) cRNA

The effect of a low dose of actinomycin D (0.1 Fg/ml) on the transcription and cel- lular distribution of virus-specific A( +) cRNA was investigated since there is evidence that this dose of actinomycin D specifically affects the function of the nucleolus (Rickinson and Dendy, 1969; Minor and Dimmock, 1977). The overall amount of virus-specific A( +) cRNA transcription was reduced in drug-treated cells, but the reduction was confined to the cytoplasmic fraction, the nuclear cRNA showing very little change at 4.5 hr pi (Table 2). A time course of A( +) cRNA accumulation showed

that the relative concentration of A(+) cRNA in the nucleus occurred between 2 and 4 hr pi. This dose of actinomycin D pre- vented the amplification of M protein synthesis late in infection and also the production of the HA and NA proteins (Minor and Dimmock, 19’7’7; unpublished observations). Segment analysis of the virus-specific A( +) cRNA isolated from actinomycin D-treated cells is presented in Fig. 4. Early in infection the A(+) cRNA in the cytoplasm was inversely proportional to the size of the transcribed gene, i.e., there was less A(+) cRNA for the larger genes and more A(+) cRNA for the smaller genes. Later in infection the A( +> cRNAs complementary to the HA and M genes were reduced in the cytoplasm. Analysis of the relative proportions of the genome segments showed that at 4 hr pi there was a 50% reduction in gene transcripts 4 and 7 (coding for HA and M proteins), but no change in the other

TABLE 2

ESTIMATED NUMBER OF A(+) cRNA GENOME

COPIES IN THE NUCLEUS AND CYTOPLASM OF NORMAL AND ACTINOMYCIN D-TREATED CELLS AT 4.5 HR POSTINFECTION

Untreated + Actinomycin D

(0.1 pgiml)

Cytoplasm Nucleus Total

1939 138 2077

75 114 189

BARRETT, WOLSTENHOLME, AND MAHY

HOURS POST-INFECTION

I 1 3 4

HOURS POST-INFECTION

FIG. 4. Estimation of the amount of A(+) cRNA complementary to individual vRNA segments in polyadenylated RNA extracted from the nucleus and cytoplasm of FPV-infected CEF cells treated with 0.1 pg/ml actinomycin D. The values are expressed as pg A(+) cRNA/pg RNA in the extracted cell fraction. 0, P (genes l-3); 0, HA (gene 4); A, NP (gene 5); n , NA (gene 6); q , M (gene 7); A, NS (gene 8).

gene transcripts (Table 3). This correlates with the relative amounts of the various proteins found in tivo at this time. The A( +) cRNA complementary to the P genes also decreased in amount. In the nucleus, however, there was a great increase in the amount of all of the nuclear A(+) cRNAs between 2 and 4 hr pi (Fig. 4) and no altera- tion in the relative proportions of the gene transcripts (Table 3).

Effect of Cycloheximide on A(+) cRNA Accumulation

In the presence of 100 pug/ml cycloheximide the amount of A(+) cRNA was greatly reduced. A time course of infection in the presence of cycloheximide showed that A(+) cRNA accumulates slowly in the cytoplasm over the period studied. Segment analysis of the A(+) cRNA isolated from

the cytoplasm of cycloheximide treated cells showed that the accumulation of each of the A(+) cRNA segments was approximately

TABLE 3

PROPORTIONS OF INFLUENZA VIRUS A(+) cRNA TRANSCRIPTS IN CONTROL AND ACTINOMYCIN D-TREATED CELLS AT~HRPOSTINFECTION

Cytoplasm Nucleus

Control + Actinomycin D + actinomycin D

1-3” 1.0 1.0 1.0 4 4.0 1.9 4.9

5 7.9 10.4 7.5

6 11.6 11.2 11.1 7 33.6 18.1 27.7 8 38.4 35.6 33.9

n Molar ratio of copies of each gene is expressed relative to the P genes which are given a value of 1.0.


:

NUCLEUS

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L I , I I 1 I I I I 1 I 2 3 4 I 1 3 4

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FIG. 5. Estimation of the amount of A(+) cRNA complementary to individual vRNA segments in polyadenylated RNA extracted from the nucleus and cytoplasm of FPV-infected CEF cells treated with 100 pg/ml cycloheximide. The values are expressed as pg A(+) cRNA/pg RNA in the extracted cell fraction. 0, P (genes l-3); 0, HA (gene 4); A, NP (gene 5); n , NA (gene 6); q , M (gene 7); A, NS (gene 8).

linear, with no evidence of transcriptional control. There was an increase in the A(+) cRNA in the nucleus up to 2 hr pi and then a decline (see Fig. 5).

Accumulation of A(-) cRNA and vRNA in infected cells

The nonpolyadenylated RNA fraction from infected cells was extracted at various times after infection and the virus-specific RNA measured as described above. The results are shown in Fig. 6. Virus-specific A(-) cRNA was detected almost immediately, but the amounts present increased dramatically between 1.5 and 2.5 hr pi. Af- ter 3 hr pi there was little or no increase in the amounts of A(-) cRNA in infected cells. The amount of vRNA remained at the input level (approximately 60 copies/cell) for the first hour after infection, increased slowly at first, then at a faster rate from 2.5 hr pi. From these results it appears that

FIG. 6. Estimation of the number of genome copies of A(-) cRNA and vRNA in the nucleus and cytoplasm of FPV-infected cells at various times postinfection. Upper panel, A(-) cRNA; lower panel, vRNA; 0, cytoplasm; 0, nucleus.

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nonpolyadenylated cRNA is synthesized earlier in the infection than is vRNA.

Both classes of RNA were found in significant amounts in both nucleus and cytoplasm. The rate of accumulation of vRNA was similar in nucleus and cytoplasm but between 1 and 3 hr pi A( -) cRNA accumulated at a much faster rate in the cytoplasm than in the nucleus.

Effects of Actinomycin D and Cyelohex- imide on the Accumulation of A(-) cRNA and vRNA in Infected Cells

The effects of 0.1 pg/ml actinomycin D and 100 pglml cycloheximide on the accumulation of these classes of RNA were studied. The effects of actinomycin D are shown in Fig. 7. The amount of virus-specific A(-) cRNA present in actinomycin D-treated cells was similar to that in control cells 1 hr after infection, but at later times there was a twofold reduction in the nucleus and a fivefold reduction in the cytoplasm. The accumulation of vRNA was similar in both drug-treated and control cells until 2 hr pi, but the marked increase in the amounts of vRNA found after 2 hr in normal cells was abolished in both the nucleus and cytoplasm of drug-treated cells.

Cycloheximide at the concentration used restricts virus-specific RNA synthesis to that which can be performed by the input virus transcriptase. The presence of the drug allowed a small accumulation of A(-) cRNA in both cell fractions up to 2 hr pi. No increase in the total amount of vRNA could be detected (see Fig. 8).

DISCUSSION

In this paper we describe methods which can be used to measure accurately the amounts of virus-specific RNA produced in influenza-infected cells. In these cells three classes of virus-specific RNA can be detected: (i) vRNA, the virus genome RNA, and (ii) A( +) cRNA. This is not a full-length copy of the virus genome (Hay et al., 1977) but has all the characteristics of mRNA. It is capped at the 5’ end and polyadenylated at the 3’ end (Krug et al., 19’76; Glass et al., 1975). (iii) A(-) cRNA, which is not polyadenylated at the 3’ end. This is assumed

to be a full-length copy of the vRNA, and to act as a template for its synthesis.

The method used for analysis of the nonpolyadenylated RNA fraction, which contains both A(-) cRNA and vRNA, differs from that used by Taylor et al. (1977), in that instead of analyzing the results kineti- cally, we add a small amount of purified vRNA to alter the position of equilibrium of the hybridization reaction. Although this requires more hybridization reactions, the mathematical treatment of the results is considerably simplified. Another advantage of this method is that it does not depend upon assumptions concerning the relative rates of annealing between the various nucleic acid species, although it does assume that all sequences present in vRNA are present in the labeled probes. We have shown that this is in fact the case.

There is temporal control of protein synthesis in influenza virus-infected cells. Some proteins (NS, NP) are synthesized in greater amounts early in infection while others (M, HA, NA) are synthesized in greater amounts late in infection (Skehel, 1972, 1973; Inglis et al., 1976). There is evidence from cell-free translation studies that this reflects the proportion of cytoplasmic mRNAs present at different times after infection (Inglis ei al., 1978; Inglis and Mahy, 1979). By analysis of pulse-labeled RNA, Hay et al. (1977) found that the maximal rates of synthesis of the various A(+) cRNAs varied during infection, indicating differential control of their transcription: maximal rates of synthesis of the RNAs were in the order 8; 1,2, 3, and 5; 4, 6, and 7. Our results, in which the accumulated proportions of the different A(+) cRNAs were determined at different times after infection are in agreement with this order of maximal synthesis. The possibility that all genes are transcribed equally in the nucleus but are transported to the cytoplasm in varying amounts at different times after infection can be eliminated, since there were no nuclear-cytoplasmic differences in the relative abundances of any of the A(+) cRNAs at any time after infection. It is now clear that in normal infections with influenza virus there is differential control of transcription of the polyadenylated RNA species.

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The rate of accumulation of virus-specific A(-) cRNA in infected cells was similar to that reported for total cRNA by-Taylor et al. (1977) and Mark et al. (1978, 1979), in that cRNA is synthesized prior to vRNA. Immediately after infection, we consis- tently found some 60 virus genome equiva- lents per cell, representing input virus. This amount was maintained during the first hour pi before increasing steadily for at least 6 hr (Fig. 6). Hay et al. (1977) reported that the bulk of vRNA was synthesized very early in infection. However, they did not measure the amount of vRNA in infected cells directly, but pulse-labeled the cells and measured the amount of radioactivity incorporated into released virus. It is possible that much of the vRNA which accumulates in infected cells is not incorporated into progeny virus, but pulse-labeling experiments can be complicated by variations in precursor pool sizes which may also account for the different results. We found a significant amount of A(-) cRNA and vRNA in both the nucleus and the cytoplasm and our results, therefore, do not allow any con- clusions to be drawn as regards site(s) of synthesis of A(-) cRNA and vRNA.

The drugs cycloheximide and actindmycin D affected virus-specific RNA accumulation in different ways. The concentration of cycloheximide used reduces protein synthesis by at least 98% and permits only primary transcription to occur, i.e., that which can be carried out by the input virus transcriptase (Repik et al., 1974; Lamb and Choppin, 1976). Low concentrations of actinomycin D , unlike high concentrations, allow some virus transcription to occur. In the presence of low doses of actinomycin D, production of the late virus proteins is specifically inhibited (Minor and Dimmock, 1977; Inglis et aZ., 1978).

In cycloheximide-treated cells A( +) cRNA transcripts of all genes accumulated linearly in the cytoplasmic fraction. This result con- firms the observation of Inglis and Mahy (1979) that in the absence of protein synthesis virus transcription is unselective. They found that immediately after lifting a cycloheximide protein-synthesis block all virus proteins were produced in equal amounts regardless of the time pi at which

the block was lifted. A similar result was observed by Lamb and Choppin (1976), who also reported that after prolonged infection in the presence of cycloheximide they could detect some differential protein synthesis. This slight anomaly may be due either to the different virus and host cell used in their experiments, or to a small residual amount of protein synthesis in the presence of cycloheximide. It appears, therefore, that the control of influenza virus A( +) cRNA transcription is dependent on protein synthesis, very probably a virus-specific protein which can alter the polymerase specificity in some way. In the presence of cycloheximide there was a very small increase in the amount of A(-) cRNA between 1 and 2 hr pi in the cytoplasm but no increase in vRNA could be detected. The absence of vRNA is consistent with the absence of significant amounts of its template cRNA. This implies, in agreement with Hay et al. (1977), that protein synthesis is required for the production of A(-) cRNA as well as for the control of A(+) cRNA transcription.

In the actinomycin D-treated cells there was a small increase in A( +) cRNA in both the nucleus and cytoplasm up to 2 hr pi. However, between 2 and 4 hr there was a much greater increase in A( +) cRNA in the nucleus and no increase in the cytoplasm. This is the exact opposite to what happens in normal infections (Barrett e+ oz., 1978). Mark et al. (1979) also found that treatment of cells with actinomycin D (2 pg/ml) confined 80-90% of WSN strain A(+) cRNA to the nucleus. These results are consistent with a nuclear site for A(+) cRNA transcription. Low doses of actinomycin D may specifically affect the nucleolus since actinomycin D intercalates specifically between GC base pairs in DNA and the nucleolus is GC rich. The nucleolus has been impli- cated in the transport of messenger RNAs from the nucleus to the cytoplasm (Deak et al., 1972). Within the nuclear fraction, analysis of individual A(+) cRNAs synthesized in the presence of actinomycin D showed no specific reduction in any gene transcript (Table 3). A similar result was reported by Mark et al. (1979), who, however, did not examine the cytoplasmic fraction.


Analysis of individual A( +) cRNAs in the cytoplasm of actinomycin D-treated cells showed a decrease after 2 hr of the late protein (M and HA) gene A(+) cRNAs while the early protein (NP, NS) gene A(+) cRNAs increased. We believe that the 50% reduction in the proportion of gene transcripts 4 and 7 in the cytoplasm of actinomycin D-treated cells must result from a specific failure to transport these RNAs from nucleus to cytoplasm, which in turn leads to the observed block in HA and M protein synthesis. Accumulation of vRNA occurred at a normal rate in cells treated with actinomycin D until 2 hr pi, after which no further increase in vRNA accumulation could be detected. Taken to- gether, these results imply that an actinomycin D-sensitive function, perhaps nucleolar- mediated transport of virus-specific macro- molecules, occurs at about 2 hr pi in influenza-infected cells. Inhibition of this function results not only in hold-up of most of the poly A(+) cRNA in the nucleus, and cessation of vRNA synthesis, but also in specific reduction in the transport of transcripts 4 and 7, coding for late virus- specific proteins.

We are at present investigating whether the function inhibited by low doses of actinomycin D is the same as that blocked in nonpermissive host cells (Bosch et al., 1978; Valcavi et al., 1978) and in permissive cells after ultraviolet irradiation (Mahy et al., 1977; Inglis et al., 1978), in which there is a similar reduction in late protein synthesis.

ACKNOWLEDGMENTS

We are grateful to Miss Nurit Kitron for excellent technical assistance and to Dr. A. C. Minson for ad- vice on the iodination procedure. This work was sup- ported by a grant from the Medical Research Council.

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Transcription and replication of influenza virus RNA