98, 211-225 (1979)

VIROLOGY

Transcription THOMAS

BARRETT,

Division of Virology, Addenhoke’s

and Replication

ADRIAN

of Influenza

J. WOLSTENHOLME,

Virus RNA

AND BRIAN

Department of Pathology, University of Cambridge, Laboratories Hospital, Hills Road, Cambridge CB2 2QQ, England Accepted

May

W. J. MAHY’ Block,

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 virionassociated 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 fibroblasts; FPV, fowl plague virus; HA, haemagglutinin; M, matrix protein; NA, neuraminidase; NP, nucleoprotein; NS, nonstructural protein; PBS, phosphatebuffered 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 process (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 All rights

Q 19’79 by Academic Press, Inc. of reproduction in any form reserved.

212

BARRETT,

WOLSTENHOLME,

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

AND

MAHY

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, MATERIALS AND METHODS 1.5 mM MgC&, 10 mM Tris-HCI, pH 7.4) Cells and virus. Monolayer cultures of containing sodium deoxycholate (0.2% w/v) CEF cells, grown in plastic petri dishes, and Nonidet P-40 (1% v/v). After thorough were prepared as previously described mixing for 1 min the nuclei were again sepa(Borland and Mahy, 1968). Stocks of influrated by centrifugation at 1000 g for 2 min. enza A virus (FPV, Restock strain) were The supernatant was combined with the grown in 11-day fertile hen eggs; the eggs previous cytoplasmic supernatant and this were infected with approximately lo3 PFU fraction was used as the cytoplasmic fraction. of virus and the allantoic fluid was harvested The nuclear pellet was washed three after 24 hr of growth when the virus titer times by resuspension and centrifugation was greater than lo9 PFU/ml. For infection, in 0.32 M sucrose, 1 mM MgCl*, and the cells were washed in phosphate-buffered final nuclear pellet was resuspended in RSB. saline (PBS), then overlaid with this alExtraction of RNA. The nuclear and cylantoic fluid diluted 1 in 4 with PBS (ap- toplasmic fractions were each mixed with proximate m.o.i. 50 PFU/cell). After 30 min equal volumes of 1 mg/ml Pronase in buffer of adsorption at room temperature, the cells (0.05 M NaCI, 0.01 M EDTA, 0.5% SDS, were drained, overlaid with medium 199 0.1 M Tris-HCI, pH 7.5) and incubated for containing 2% calf serum, and incubated at 60 min at 37”. The aqueous phase was twice 37” (zero time). extracted at 37” with equal volumes of DmLg treatments. Confluent CEF cells chloroform-isoamylalcohol (25:l) and the were pretreated for 1 hr with medium con- final aqueous phase precipitated overnight, taining the appropriate concentration of ei- after addition of 2.5 vol ethanol, at -20”. ther actinomycin D or cycloheximide. In all The precipitated nucleic acid fractions from cases 0.1 pg/ml actinomycin D was used. the nuclei were collected by centrifugation and resuspended in 0.1 M NaCl, 0.015 M This is not sufficient to completely inhibit virus transcription but appears to affect pri- MgC&, 0.1 M Tris-HCI, pH 7.4, and incumarily host nucleolar function (Rickinson bated with DNase I (50 kg/ml) for 2 hr at and Dendy, 1969). Cycloheximide was used 28”. RNA was then purified from the mixat 100 pg/ml which is sufficient to inhibit ture by Pronase treatment, chloroformcell protein synthesis by at least 98% (Lamb isoamylalcohol extraction, and precipitation and Choppin, 1976). Virus adsorption was in ethanol at -20” as before. In some cases extraction carried out in the presence of the same con- the chloroform-isoamylalcohol centration of drug added to the virus sus- was replaced by phenol-chloroform (1:l) pension. After 30 min of adsorption at room extraction. To avoid trapping polyadenyltemperature, the cell monolayers were ated RNA in the interphase the phenol drained, overlaid with medium 199 containlayer was reextracted with 0.1 M Trising 2% calf serum and the appropriate HCl, pH 9.0, 150 mM NaCl. The RNA preamount of drug, and incubated at 37”. cipitates were then collected by centrifugaCell fractionation. At various times post- tion, dissolved in TE buffer (1 mM EDTA, infection, the medium was removed from 10 mM Tris-HCI, pH 7.4), and dimethyl the cells and replaced with ice-cold PBS. sulfoxide was added to a final concentration of 90% before heating to 45” for 20 min to The cells were harvested by scraping with denature the RNA. The RNA was reprea rubber policeman and pelleted by centrifugation at 1000 g for 2 min. The cell pellets cipitated in ethanol as before. Polyadewere resuspended in B4 buffer (10 mM nylated and nonpolyadenylated fractions of NaCl, 1.6 mM MgCl%, 1 mM triethanolthe RNA were prepared by oligo(dT)-celluamine, 10 mM Tris-HCl, pH 7.4), kept at lose chromatography. 0” for 15 min, then homogenized by 15 Separation of polyadenylated RNA. Poly-

INFLUENZA

VIRUS

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 icecold 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 modification of the method described by Commerford (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 Brownlee and Cartwright (1977) were used, since

RNA

SYNTHESIS

21X

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 intervals 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 denatured 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 virusspeci$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-

214

BARRETT,

WOLSTENHOLME,

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 virusspecije 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 -= c

Y

then c=-

x y-x

pg

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]thymidine-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 Logistics, Virology Cancer Program, Viral Oncology, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland. RESULTS

and v = xc pg,

Characteristics of Radioactively Inflzcenxa VRNA and cDNA

where v = amount of virus genome RNA, c = amount of co.mpIementary

AND MAHY

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-

Labeled

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 sufficiently resolved and were extracted as one band. When the isolated segment RNAs

‘pale1na[ta aq u’i13 aidures aq3 u! ~~83 ($ )v ay!aads styA 30 ?unoum [enlse aql ‘svN~ pa,aqe[-Iszr a9130 ,Cl!a!~ae ayyads aql BU!MOU)I ‘uoy.mles le punoq qunoa 929 = fcao.rd!saJ ‘6~00’0 = qdaa.ralu! ‘(a) p aua3 ‘uo!le.mles JE punoq swnoa pgp = Ieao.td!aaJ ‘FZ~‘O = $daa.ralu! ‘(m) sauaS d :BJB.M san[eA [en&se aq& ‘anlea uo!le.m$es aq~ 30 1caoJd:aa.c aq~ sang ajau!P~o aql uo au![ aql30 zdaaralu! aq~, @ued JJMOI) pa?$oId a.ca~ alep s!q$ 30 san1e.t Ieao.cd!sal-a[qnop san[aa uo!?a.tnqes urnuqxscu aq~ au!m.ta)ap o& .[aued raddn aq? u! pano[d a.re sl1nsa.t aq~ Pue ~uaw~ear~ aseafanu bq pa.mseacu J.GJM [aaa[ Indu! qxa hq pa/JaloJd sJunoa aq~, ‘VN~ auas-1 $ZI JeqJ 3” spInouIe %u~sea.lau! q&u 69~ a.@ 01 pa.qnhax VN~ 11a3 (+)v 30 lunoum uv ‘(a) p aua8 pa[aqe[-Ipa, pwp!Jqqhq uaw s&+i VNX auaWZi q 58a 30 urda 0001 ql!~ uo!vz!pyqhq 03 paz!p!JqLq set yd ~q T vN~ (+)v ~&use~do&Ca ‘lau-ed ~q%!x ‘snapnu Jad satdoa auaS vN81a ~29 pue sna[anu .rad PW (H) sauaz d PaIaqwpZr sardoa aual vNx3 (-)v ozp SE pajs[nyt?” aq w?a s!ql uo!~aeq [Ia> Lad saidos aua% 30 slural UI %I PE’O = o put? 74 82.0 = 3 leql al%qna[en ue3 am suoymha asaql %u!sn 75.1 = 3/(z + fi) 103 pue 61’~ = 3/n ~03 anIe.4 aql 826 s! vNxh-IFZ, q+k5 pau!elqo aq u83 q9iqM uoyz!p!1qhq umuqxrrm ayl aau!S ‘801; SBM anleA aq? VN~ [[aa 30 we&o,m!m Jad VNHI” pappe Bu z 30 aauasald aql UI ‘aaw+sar astFa[anu y,og p um!.rq![~nha paqwaz uo~y 02 paZ!pyqn’q uaw ‘~&)()I se.~ an[‘eA umyq!I!nba aqq VN~A p~uo!~pp~ 30 aauasqe pw -38~~ aq9 VNXA pawe 30 -we aw u! vNx~-br ‘(spoq?am pue s[“yal~~ aas) wseldoQa aauasard aql u! tfNaa[HJ 03 paZ!p!Jqkq UaqM ‘!d .rq p ‘vN8 pale@uapeQoduou .ma~XIu ‘slaued ~aya3 dad sa!doa auai? ~NHA 1~1 pue wseldo$ Jad sa!doa JUJ8 vNxa (-)v 8~~1 se pa?e[na[ea aq uea srq$ uoy~13 [I~J Lad saldoa aua8 30 stuJa3 UI -%vi ~9.0 = n puz Srf 9b.p = 3 leyl a~epwp?a uea a~ suo!Jsnba asaql 8u~sn ‘~‘0 = J/(Z + O) aio3aJaql ‘aaue?s!saJ aseapnu sps p paqaeaJ SF ump -q!Iinba ‘VN~ i[aa 30 un&o~s!tu Lad VN~A pappe 30 8u ~30 aauasa.td aq$ UI .zl‘(j = ajrl a.I03aJaq? ‘y&l SBM anPU. rungqII!nha Jql VN~A pappe 30 J3”JSqV aq? U! VNa”[&] 0% pJZ!p!.K@q UJqM ‘(L&I ‘.lTl 38 AJ(.bJ, ‘h[EJUUe obo()I 01 lUJ[lLt!dJJ) VNxIa pJppE 30 WCWq7: PUE aauasa.td aq’J u! 0$96-~ amM ~N~A-~~~~ 03 Paz!P!JqLq uaq~ satqR4 umpq![!nh3 yd 1q z ‘vN~ pale[huapaQoduou s!Luse[do?ba ‘[auvd ~a? :SMOI[OJ se sm s?psaJ JO uoy~eplJ[e~ ‘J~N~A-I~~, 0~ VN~ pa7yduapeQod [[aJ pJ~3JJU! 30 UO~~~Zp.Kp& :Iaued Jq8!8 ‘VNH [Ia2 30 we.L?O.K+1 Jad V~8.t ,\dJ paIaqelTm 30 %U z 30 (0) aauasqe pue (0) aauasald aq? u! pawo3Jad a.IaM suorJez!p!.Iqbq asea qasa UI .(slarred .raMol) ~N~A-I’.~, pu” (slauvd Jaddn) vNa&.] 07 VN~ %U!Stl VNg cn3!>ads-sn.qa 30 S!Sz@U~ ‘1 ‘OId pay+Cuapebloduou [[aa palaaju! 30 uo!lezlp!JqiCH :sIaued a[pp!w pue 73a7 ‘vN~.~-&, p UB VNa3[Ha]

1

oz

ot

09

08

216

BARRETT,

WOLSTENHOLME,

AND MAHY

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 digestion 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

FIG. 2. Polyacrylamide gel electrophoresis of 9labeled 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.

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 .

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 sequence analysis (Robertson, 1979). The intrinsic 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

z “-

L-I 16’

IO+

10-I

IO0

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

INFLUENZA

TABLE PROPORTIONS

1.5

2.5

1.0

1.0

1.5 4.3 2.7 7.4 9.9

3.5 8.7

Gene No.

1.0

1-3” 4 5 6 8

1

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

AND CYTOPLASM

Nucleus (hr postinfection)

Cytoplasm (hr postinfection)

7

217

VIRUS RNA SYNTHESIS

4.5

1.5

1.0

1.0

6.1 18.8

5.2 9.5

1.0 1.8

7.1

7.0

9.5

12.9 27.3

15.9 30.5

24.3 27.7

2.8 2.4 5.2 10.8

2.5

4.5

Expected ratio”

1.0

1.0

1.0

2.9 8.9 7.9 17.8 32.6

7.6 7.5 8.3 25.5 26.5

1.3 1.6 1.9

3.7 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 accumulating 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 cellular 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 prevented 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 (0.1 pgiml)

Cytoplasm

Nucleus

Total

1939

138

2077

75

114

189

D

BARRETT,

WOLSTENHOLME,

AND MAHY

I HOURS

POST-INFECTION

1 HOURS

4

3 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 alteration in the relative proportions of the gene transcripts (Table 3). Effect of Cycloheximide Accumulation

on A(+)

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

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

cRNA

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

3

1-3” 4 5 6

7 8

1.0 4.0 7.9 11.6 33.6 38.4

+ Actinomycin 1.0 1.9 10.4 11.2

18.1 35.6

D

Nucleus + actinomycin D 1.0 4.9 7.5

11.1 27.7 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.

INFLUENZA

219

VIRUS RNA SYNTHESIS

NUCLEUS

A.’

CRNA

:

L

I

,

I

2 “OURS

3

I

I

4

1

I

I

I

I

1

3

HOURS

POST-INECIION

I

1

4

POSl-INFECIION

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. After 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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FIG. ‘7. Estimation of the number of genome copies of A(-) cRNA and vRNA in the nucleus and cytoplasm of FPV-infected cells in the presence and absence of actinomycin D. 0, Untreated cells; 0, cells treated with 0.1 Fg/ml actinomycin D. Upper left panel, nucleus A(-) cRNA, lower left panel, cytoplasm A(-) cRNA. Upper right panel, nucleus vRNA; lower right panel, cytoplasm vRNA.

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VIRUS

RNA

SYNTHESIS

221

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 kinetically, 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 Effects of Actinomycin D and Cyelohexmathematical treatment of the results is imide on the Accumulation of A(-) considerably simplified. Another advantage cRNA and vRNA in Infected Cells The effects of 0.1 pg/ml actinomycin D of this method is that it does not depend upon assumptions concerning the relative and 100 pglml cycloheximide on the accumurates of annealing between the various nulation of these classes of RNA were studied. cleic acid species, although it does assume The effects of actinomycin D are shown in that all sequences present in vRNA are Fig. 7. The amount of virus-specific A(-) present in the labeled probes. We have cRNA present in actinomycin D-treated cells was similar to that in control cells 1 hr shown that this is in fact the case. There is temporal control of protein synafter infection, but at later times there was a twofold reduction in the nucleus and a thesis in influenza virus-infected cells. Some in fivefold reduction in the cytoplasm. The ac- proteins (NS, NP) are synthesized greater amounts early in infection while cumulation of vRNA was similar in both drug-treated and control cells until 2 hr pi, others (M, HA, NA) are synthesized in but the marked increase in the amounts of greater amounts late in infection (Skehel, vRNA found after 2 hr in normal cells was 1972, 1973; Inglis et al., 1976). There is evidence from cell-free translation studies that abolished in both the nucleus and cytoplasm this reflects the proportion of cytoplasmic of drug-treated cells. mRNAs present at different times after inCycloheximide at the concentration used fection (Inglis ei al., 1978; Inglis and Mahy, restricts virus-specific RNA synthesis to 1979). By analysis of pulse-labeled RNA, that which can be performed by the input Hay et al. (1977) found that the maximal virus transcriptase. The presence of the rates of synthesis of the various A(+) drug allowed a small accumulation of A(-) cRNAs varied during infection, indicating cRNA in both cell fractions up to 2 hr pi. differential control of their transcription: No increase in the total amount of vRNA maximal rates of synthesis of the RNAs could be detected (see Fig. 8). were in the order 8; 1,2, 3, and 5; 4, 6, and 7. Our results, in which the accumulated DISCUSSION proportions of the different A(+) cRNAs In this paper we describe methods which were determined at different times after can be used to measure accurately the infection are in agreement with this order of amounts of virus-specific RNA produced in maximal synthesis. The possibility that all influenza-infected cells. In these cells three genes are transcribed equally in the nucleus classes of virus-specific RNA can be de- but are transported to the cytoplasm in tected: (i) vRNA, the virus genome RNA, varying amounts at different times after inand (ii) A( +) cRNA. This is not a full-length fection can be eliminated, since there were copy of the virus genome (Hay et al., 1977) no nuclear-cytoplasmic differences in the but has all the characteristics of mRNA. relative abundances of any of the A(+) It is capped at the 5’ end and polyadenylated cRNAs at any time after infection. It is now at the 3’ end (Krug et al., 19’76; Glass et al., clear that in normal infections with influenza 1975). (iii) A(-) cRNA, which is not poly- virus there is differential control of tranadenylated at the 3’ end. This is assumed scription of the polyadenylated RNA species. 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.

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FIG. 8. Estimation of the number of genome copies of A( -) cRNA and vRNA in the nucleus and cytoplasm of FPV-infected and absence of cycloheximide. 0, Untreated cells; 0, cells treated with 100 Fglml cycloheximide. Upper left panel, nucleus panel, cytoplasm A(-) cRNA; upper right panel, nucleus vRNA; lower right panel, cytoplasm vRNA.

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cells in the presence A( -) cRNA; lower left

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INFLUENZA

VIRUS RNA SYNTHESIS

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 consistently found some 60 virus genome equivalents 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 conclusions 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 confirms 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

223

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 implicated 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.

224

BARRETT,

WOLSTENHOLME,

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 together, these results imply that an actinomycin D-sensitive function, perhaps nucleolarmediated transport of virus-specific macromolecules, 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 virusspecific 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 advice on the iodination procedure. This work was supported by a grant from the Medical Research Council. REFERENCES ARMSTRONG, S. J., and BARRY, R. D. (19’74). The topography of RNA synthesis in cells infected with fowl plague virus. J. Gen. Viral. 24, 535-547. BARRETT, T., BROWNSON, J. M., WOLSTENHOLME, A. J., and MAHY, B. W. J. (1978). Studies on the synthesis of cRNA and vRNA in cells infected with influenza virus. In “Negative Strand Viruses and the Host Cell” (B. W. J. Mahy and R. D. Barry,

AND MAHY

eds.), pp. 325-331. Academic Press, London/New York. BARRY, R. D., and MAHY, B. W. J. (1979). The influenza virus genome and its replication. Brit. Med. Bull. 35, 39-46. BORLAND, R., and MAHY, B. W. J. (1968). Deoxyribonucleic acid-dependent ribonucleic acid polymerase activity in cells infected with influenza virus. J. Viral. 2, 33-39. BOSCH, F. X., HAY, A. J., and SKEHEL, J. J. (1978). RNA and protein synthesis in a permissive and an abortive influenza virus infection. In “Negative Strand Viruses and the Host Cell” (B. W. J. Mahy and R. D. Barry, eds.), pp. 465-474. Academic Press, London/New York. BROWNLEE, G. G., and CARTWRIGHT, E. M. (1977). Rapid gel sequencing of RNA by primed synthesis with reverse transcriptase. J. Mol. Biol. 114, 93-117. COMMERFORD, S. L. (1971). Iodination of nucleic acids in vitro. Biochemistry 10, 1993-2000. DEAK, I., SIDEBOTTOM, E., and HARRIS, H. (1972). Further experiments on the role of the nucleolus in the expression of structural genes. J. Cell Sci. 11, 379-391. FOLLETT, E. A. C., PRINGLE, C. R., WUNNER, W. H., and SKEHEL, J. J. (1974). Virus replication in enucleate cells: Vesicular stomatitis virus and influenza virus. J. Viral. 13, 394-399. GLASS, S. E., MCGEOCH, D., and BARRY, R. D. (1975). Characterisation of the mRNA of influenza virus. J. Vi&. 16, 1435-1443. HAY, A. J., LOMNICZI, B., BELLAMY, A. R., and SKEHEL, J. J. (1977). Transcription of the influenza virus genome. Virology 83, 337-355. INGLIS, S. C., CARROLL, A. R., LAMB, R. A., and MAHY, B. W. J. (1976). Polypeptides specified by the influenza virus genome. 1. Evidence for eight distinct gene products specified by fowl plague virus. Virology 74, 489-503. INGLIS, S. C., CONTI, G., and MAHY, B. W. J. (1978). Control of influenza virus polypeptide synthesis. In “Negative Strand Viruses and the Host Cell” (B. W. J. Mahy and R. D. Barry, eds.), pp. 239-248. Academic Press, London/New York. INGLIS, S. C., and MAHY, B. W. J. (1979). Polypeptides specified by the influenza virus genome. 3. Control of synthesis in infected cells. Virology 95, 154164. JEPPESEN, P. G. N., BARRELL, B. G., SANGER, F., and COULSON, A. R. (1972). Nucleotide sequences of two fragments from the coat-protein cistron of bacteriophage R17 ribonucleic acid. Biochem. J. 128, 993-1006. 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. 13,1156-1161. KRUG, R. M., MORGAN, M. A., and SHATKIN, A. J.

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(1976). Influenza viral mRNA contains internal N6methyladenosine and 5’-terminal7-methylguanosine in cap structures. J. Viral. 20, 45-53. LAMB, R. A., and CHOPPIN, P. W. (1976). Synthesis of influenza virus proteins in infected cells: translation of viral polypeptides, including three P polypeptides, from RNA produced by primary transcription. Virology 74, 504-519. MCGEOCH, D., FELLNER, P., and NEWTON, C. (1976). Influenza virus genome consists of eight distinct RNA species. Proc. Nat. Acad. Sci. USA 73,30453049.

MAHY, B. W. J., CARROLL, A. R., BROWNSON, J. M. T., and MCGEOCH, D. J. (1977). Block to influenza virus replication in cells preirradiated with ultraviolet light. Virology 83, 150-162. MARK, G. E., TAYLOR, J. M., HERRING, L., BRONI, B., and KRUG, R. M. (1978). Transcription and replication of the influenza virus genome early after infection. In “Negative Strand Viruses and the Host Cell” (B. W. J. Mahy and R. D. Barry, eds.), pp. 333-340. Academic Press, London/New York. MARK, G. E., TAYLOR, J. M., BRONI, B., and KRUG, R. M. (1979). Nuclear accumulation of influenza viral RNA transcripts and the effects of cycloheximide, actinomycin D, and a-amanitin. J. Viral. 29, 744752. MINOR, P. D., and DIMMOCK, N. J. (1975). Inhibition of synthesis of influenza virus proteins: Evidence for

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two host-cell-dependent events during multiplication. Virology 67, 114-123. MINOR, P. D., and DIMMOCK, N. J. (1977). Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function. Virology 78, 393-406. REPIK, P., FLAMMAND, A., and BISHOP, D. H. L. (1974). Effect of interferon upon the primary and secondary transcription of vesicular stomatitis virus. J. Virol. 14, 1169-1178. RICKINSON, A. B., and DENDY, P. P. (1969). The use of actinomycin D in studies of nucleolar function. Ezperientia 25, 1251-1253. ROBERTSON, J. S. (1979). Nucleotide sequences from the terminal regions of fowl plague virus genome RNA. Proc. Roy. Sot. Ser. B, in press. SKEHEL, J. J. (1972). Polypeptide synthesis in influenza virus-infected cells. Virology 49, 23-36. SKEHEL, J. J. (19’73). Early polypeptide synthesis in influenza virus-infected cells. Virology 56, 394-399. TAYLOR, J. M., ILLMENSEE, R., LITWIN, S., HERRING, L., BRONI, B., and KRUG, R. M. (1977). Use of specific radioactive probes to study transcription and replication of the influenza virus genome. J. Viral. 21, 530-540. VALCAVI, P., CONTI, G., and SCHITO, G. C. (1978). Macromolecular synthesis during abortive infection of KB cells by influenza virus. In “Negative Strand Viruses and the Host Cell” (B. W. J. Mahy and R. D. Barry, eds.), pp. 475-481. Academic Press, London/New York.

Transcription and replication of influenza virus RNA.

98, 211-225 (1979) VIROLOGY Transcription THOMAS BARRETT, Division of Virology, Addenhoke’s and Replication ADRIAN of Influenza J. WOLSTENHOLM...
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