Polypeptides specified by the influenza virus genome

VIROLOGY

74, 489403

(1976)

Polypeptides I. Evidence

for Eight

STEPHEN

Division

C. INGLIS,’

of Virology,

Specified Distinct

by the Influenza

Gene Products

ANTHONY BRIAN

Specified

R. CARROLL,2 W. J. MAHY

Department ofPathology, University of Cambridge, Hospital, Hills Road, Cambridge CB2 2QQ, Accepted

June

Virus Genome by Fowl

ROBERT

Laboratories England

Plague

Virus

A. LAMB,”

Block,

AND

Addenbrooke’s

II,1976

The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis. We detected eight distinct virus gene products: three polymerase-associated polypeptides (P,, P,, P,), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), membrane polypeptide (M), and a nonstructural polypeptide (NS). The molecular weights of these polypeptides correlate closely with the molecular weights of the eight genome RNA species found in fowl plague virus. The three high molecular weight polypeptides, P,, P,, and P,, were detected both in virions and infected cells, and their separate identity established by a two-dimensional tryptic peptide mapping procedure. An active RNA polymerase enzyme complex isolated from virions and virus-infected cells contained all three P proteins together with the NP protein. The nonstructural polypeptide (NS), together with the P proteins and the NP, appeared early in the infectious cycle, while the M protein and HA protein appeared later in infection. The NS and M polypeptides, which have similar molecular weights, were separated on SDS-polyacrylamide gels and shown to be distinct by tryptic peptide mapping.

hemagglutinin (HA), which is normally cleaved in the mature virion into two smaller polypeptides, (HA, and HA,); the neuraminidase (NA); the membrane protein (M); the nucleoprotein (NP); and a high molecular weight protein (PI, thought to be associated with RNA polymerase activity of the virion. There is general agreement between various laboratories as to the size and number of these polypeptides in the influenza virion (White, 19741, with the exception of the P protein. This constitutes only about 3% of the total virus protein, but whereas some laboratories find only one P polypeptide (Compans et al., 1970; Schulze, 1970; Klenk et al., 1972), others report two (Skehe1 and Schild, 1971; Hefti et al., 1975). In addition to the virion structural polypeptides, influenza virus-infected cells have been reported to contain a virus-in-

INTRODUCTION

Influenza viruses of several different strains, grown in eggs or in tissue culture, have been found to contain at least seven species of polypeptide when analyzed by SDS*-polyacrylamide gel electrophoresis (Cornpans et aZ., 1970; Skehel and Schild, 1971; Skehel, 1972). These consist of the ’ Author to whom requests for reprints should be addressed. s Present address: Department of Microbiology, The University of Virginia, School of Medicine, Charlottesville, Virginia 22901. ‘I Present address: The Rockefeller University, New York, New York 10021. ’ The following abbreviations are used: CEF, chick embryo fibroblasts; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulphate; PBS, phosphate-buffered saline; PEG, polyethylene glycol 6000; PFU, plaque-forming units; tic, thinlayer chromatography. 489 Copyright All rights

0 of

by Academic Press, Inc. reproduction in any form reserved. 1976

490

INGLIS

duced nonstructural (NS) protein of molecular weight approximately 23,000, which migrates slightly ahead of the virus structural M protein during electrophoresis on SDS-polyacrylamide gels (Dimmock and Watson, 1969; Lazarowitz et al., 1971; Skehel, 1972, 1973). Recently, the number and size of the RNA species comprising the influenza virus genome have been reevaluated by high resolution polyacrylamide-urea gel electrophoresis (McGeoch et al., 1976; Pons, 1976). These studies indicate that influenza A viruses contain eight distinct genome RNA pieces with a total molecular weight of approximately 5.9 x 106. In the present study, we analyze the structural and nonstructural polypeptides of influenza virus by high resolution polyacrylamide slab-gel electrophoresis and present evidence for eight influenza virus gene products. We find the M and NS polypeptides to be distinct as judged by their appearance at different times in the infected cell, and by two-dimensional separation of their [““Slmethionine-labeled tryptic peptides. We also show that three high molecular weight P polypeptides are associated with influenza virus transcriptase activity and are present both in influenza virions and in infected cells. MATERIALS

AND

METHODS

Cells and virus. Influenza A virus (fowl plague, Restock strain) was grown in fertile hen eggs and assayed by hemagglutination or plaque titration as previously described (Borland and Mahy, 1968). CEF cell cultures, prepared as previously described (Borland and Mahy, 19681, were infected with freshly harvested infected allantoic fluid diluted in saline to give a multiplicity of approximately 20 PFU per cell. After 30 min adsorption at room temperature the inoculum was replaced by maintenance medium (199 containing 2% calf serum) and the cultures were incubated at 37” (Zero time). Virus purification. Infected cell medium, normally harvested 24 hr postinfection, was clarified by centrifugation at 10,000 g for 10 min at 4” in an IEC B20 centrifuge. Virus was then pelleted by cen-

ET

AL.

trifugation of the clarified medium at 40,000 g in an IEC A54 rotor for 90 min at 4”. The pellet was resuspended in NTE buffer (10 mM Tris-HCl (pH 7.4); 100 mM NaCl; 1 mM EDTA) using a Dounce-type homogenizer and limited sonication (10 set) in a Dawe sonic bath, then layered onto a discontinuous sucrose gradient containing 5 ml 60% (w/v) overlaid with 5 ml of 15% (w/v) sucrose in NTE. The gradient was centrifuged at 65,000 g in an IEC SB 110 rotor for 90 min at 4”. Virus-containing fractions were pooled, diluted with an equal volume of NTE, layered onto a preformed linear gradient of 20-45% (w/v) potassium tartrate, and centrifuged to equilibrium at 180,000 g for 16 hr in an IEC SB283 rotor. Virus-containing fractions were pooled, diluted, and pelleted in an IEC A237 rotor at 180,OOOg for 30 min. The pellet was allowed to resuspend in NT buffer (10 m&f Tris-HCl, pH 7.4; 100 mM NaCl) overnight at 0”. Y3-methionine-labeled influenza virus. Confluent CEF monolayers were infected with influenza virus at an input multiplicity of 1 PFU/cell, and after 30-min adsorption the cell sheet was overlaid with Eagle’s medium (BME), methionlll, rrce. After 2 hr at 37” the medium was supplemented with 50 &i/ml of [““Slmethionine and the cultures were incubated at 37” for a further 20 hr. 13Slmethionine-labeled virus was purified from the culture medium as described above. ““S-methionine labeling of infected cells. For pulse-labeling, at different times after infection, the maintenance medium was removed, the cell sheet rinsed with PBS, and overlaid with BME (methionine-free) containing 5 &i/ml of [YSlmethionine. After 15 min incubation at 37” the labeled medium was removed and the cells were harvested into ice-cold 0.9% NaCl, collected by centrifugation, and washed again by suspension in 0.9% NaCl and recentrifugation. The cell pellets were resuspended in an appropriate volume (normally 0.2 ml for the cells from a 5-cm petri dish culture) of electrophoresis sample buffer (Laemmli, 1970) and heated at 100 for 3 min before analysis by polyacrylamide gel electrophoresis.

INFLUENZA

VIRUS

When peptide maps of the separated polypeptides were to be prepared, the amount and duration of labeling was increased as described in Results. Polyacrylamide gel electrophoresis. Virus polypeptides were analyzed by highresolution discontinuous polyacrylamide gel electrophoresis using Tris-glycine buffer and SDS (Laemmli, 1970). Slab gels were cast between two glass plates separated by 2-mm Perspex spacers and electrophoresed in an apparatus supplied by Raven Scientific Ltd., Haverhill, Suffolk, England. Samples were prepared in 6.25 r&4 Tris-HCl, pH 7.2, and 20% glycerol. Prior to analysis, SDS, dithiothreitol, and bromophenol blue were added to the samples to final concentrations of 2, 2, and 0.0050/o, respectively. Normally, 11 or 15% gels overlaid with a 5% stacking gel were used, but for resolution of high molecular weight polypeptides, the running gel concentration was reduced to 7.5%. Electrophoresis was for 16 hr at a constant voltage of 50 V, following which the gel was fixed in 50% methanol:lO% acetic acid, stained in a solution of 0.1% Coomassie brilliant blue in 10% methanol:7% acetic acid, then destained in the same solvent at 60”. The destained gel was dried under vacuum on to Whatman 3MM chromatography paper and autoradiographed using Kodak Blue Brand BB54 X-ray film. Peptide mapping of virus polypeptides. Bands containing virus polypeptides were excised from stained, dried, autoradiographed gels, and rehydrated in 1% ammonium bicarbonate. Trypsin was added to 10 pg/ml and the gel fragments shaken at 37” for 16 hr. The eluted peptides were freeze-dried, redissolved in water, and applied to a 6 x 0.5-cm Biogel P2 column to remove salt contamination. The column was eluted with water, and the labeled peptide-containing fraction dried. The peptides were redissolved in a suitable small volume of water, and samples (approximately 20,000 cpm in 5 ~1) applied to 20- x 20-cm precoated cellulose tic plates. The peptides were subjected to electrophoresis in the first dimension at 300 V for 3 hr in pH 6.4 electrophoresis buffer (pyridine: acetic acid:water, 200:8:1800), followed by

GENE

PRODUCTS

491

ascending chromatography in the second dimension in butanol:acetic acid:water: pyridine (30:6:24:20). Labeled peptides were detected by autoradiography as described for dried gels. Isolation of RNA-dependent RNA polymerase activity. A RNA-dependent RNA polymerase-active fraction from the cytoplasm of fowl plague virus-infected cells was isolated using a similar procedure to Compans and Caliguiri (1973). CEF cells were labeled with 0.4 &i/ml of [““Slmethionine from 3 to 6 hr after infection with fowl plague virus. At 6 hr postinfection the cells were harvested in ice-cold PBS and cytoplasmic fraction isolated as previously described (Hastie and Mahy, 1973) and fractionated on a discontinuous sucrose density gradient (Compans and Caliguiri, 1973). The cytoplasm was mixed with an equal volume of 6% (w/w) sucrose in RSB (10 n&f Tris-HCl, pH 7.4; 10 mM KCl; 1.5 mM MgCl,) and included in a discontinuous sucrose gradient of 3 ml of RSB, 7 ml of 25%, 10 ml of 30% (sample), 7 ml of 40%, 7 ml of 45%, and 3 ml of 60% sucrose concentrations (w/w) in RSB. The gradient was centrifuged at 70,000 g for 17 hr in a Beckman SW 27 rotor. The visible bands were collected using a syringe with a bent needle and assayed in a standard RNA polymerase reaction (Carroll et al., 1975) containing 20 pg/ml of actinomycin D. The major polymerase-containing fraction was dialyzed against B2 buffer (10 mM TrisHCI, pH 7.4; 10 mM NaCl; 1.5 mM MgCI,) overnight, then against PEG to reduce the volume to approximately 0.5 ml. This sample was layered on to a 15-30% (w/v) sucrose density gradient in NTE (12 ml) and centrifuged at 180,000 g for 2 hr at 4”. The gradient was fractionated using an ISCO fractionator and each fraction was assayed for RNA polymerase activity as before. The polymerase complex sedimented just ahead of the larger ribosomal subunit. Materials. L-[““Slmethionine (45-150 Ci/ mmol) was obtained from the Radiochemical Centre, Amersham, England. Biogel P2 was obtained from Biorad Inc., Richmond, Calif. Eagle’s BME medium (methionine-free) was obtained from Biocult Ltd., Paisley, Scotland. Acrylamide (spe-

492

INGLIS

cially purified) and bisacrylamide were obtained from British Drug Houses Ltd., Poole, Dorset. N,iV,N’,N’,tetramethylethylene diamine was obtained from Eastman Kodak, Rochester, New York. PEG was obtained from Hopkins and Williams Ltd., London, England. Polygram precoated cellulose tic plates were obtained from Macherey-Nagel & Co., Duren, Germany. RESULTS

The Structural Polypeptides of Influenza (Fowl Plague) Virus Figure la shows the polypeptide pattern of fowl plague virus grown in eggs and purified from allantoic fluid as described in Methods and Materials. The polypeptides are designated where possible by the convention adopted by Kilbourne et al. (1972). The pattern obtained is in good agreement with previously published patterns of fowl plague virus polypeptides (Klenk et al., 1972; Skehel, 1972) and the following points can be noted: (i) fowl plague virus does not contain uncleaved HA; (ii) the NP and M polypeptides are major components comprising about 60% of the total stained protein; (iii) the NA polypeptide is only a minor component; (iv) two high molecular weight P proteins

PPaP

HA

ET AL.

are resolved on 11% gels, confirming the observations of Skehel (1972). However, further analysis using 7.5% gels showed that the P, band could be resolved into two components (Fig. 2). The higher molecular weight component is designated P, and the lower P,. The results of other experiments (data not shown) indicated that only polypeptides HA,, NA, and HA, could be stained with Schifs periodate reagent or labeled with radioactive carbohydrate precursors and so were glycoproteins, and that only these three polypeptides were removed from the virion by treatment with bromelain, confirming previous reports (Cornpans et al., 1970; Brand and Skehel, 1972). Since [3”Slmethionine was the aminoacid precursor to be used in peptide mapping experiments, we compared the polypeptide pattern of purified ““S-labeled fowl plague virus obtained by autoradiography (Fig. lb) with that observed in stained gels. The pattern obtained by either method was essentially the same (cf. Fig. la and b). The dye Coomassie brilliant blue is thought to bind stoichiometrically to protein (Gibson and Roizman, 1974) whereas incorporation of methionine is obviously dependent on the number of methionine

M

( 2 ,

FIG. 1. Polyacrylamide slab gel electrophoresis of (a) unlabeled and (b) [35Slmethionine-labeled tides of purified fowl plague virus. Densitometer tracings of (a) Coomassie brilliant blue-stained an autoradiogram. Virions were grown in CEF cells. Labeling and preparation of polypeptides phoresis on 11% polyacrylamide gels were carried out as described in Materials and Methods. from left to right.

polypepgel, and (b) for electroMigration is

INFLUENZA

VIRUS

residues in each polypeptide. The relative abundances of the polypeptides, determined by densitometer scanning of a stained 9% gel or a :‘“S-autoradiogram, were similar (Table 11, and it was concluded that [YSlmethionine was a suitable labeled precursor amino-acid for the later studies. Separation of the polypeptides P, and P:, is not achieved using this gel concentration, but it is apparent that the ratio of peak P, to peak P2,:I is 1:2. When polypeptides P, and P:, were separated on a lower percentage gel (polypeptides HA, and M then migrated at the dye front), the ratio of the three peaks P,:P,:P,, was 1:l:l

P* 11 I

I

I

p3

FIG. 2. Polyacrylamide slab gel electrophoresis of high molecular weight polypeptides of purified fowl plague virus. Densitometer tracing of Coomassie brilliant blue-stained gel. Virions were grown in CEF cells, and prepared for electrophoresis as in Fig. 1, except that the polyacrylamide gel concentration was 7.5%. Migration is from left to right.

TABLE ESTIMATED

POlyjZP-

P, P2 PY NP HA, NA HA, M

NUMBER

Molecular

AND

weight”

MOLECULAR

(daltons

WEIGHTS x 10m3)

7% gel

9% gel

11% gel

95.5 87.1 85.1 53.7 47.9 44.7 -

95.5 87.1 53.1 44.7 42.7 26.6 24.0

96.6

41.7 38.9 26.9

25.7

GENE

493

PRODUCTS

as estimated by percentage stain bound (Fig. 2). The molecular weights of the virus polypeptides were estimated from the rates of migration during electrophoresis along with marker polypeptides of known molecular weight (Weber and Osborn, 1969). The estimated molecular weights for each polypeptide were similar for the three gel concentrations used, although small variations were apparent (Table 1). The molecular weights calculated for fowl plague virus polypeptides are in good agreement with previous estimates (White, 1974). The estimated molecular weights of the polypeptides not previously described were Pp, 87,000, and P:(, 85,000. The numbers of molecules of each polypeptide per virion were calculated using data from stained preparations (Table 1). Two methods of calculation were used. First, a value of 2.1 x 10Hdaltons of protein per virion was used. This value was derived from estimates of the mean particle weight of influenza viruses of 2.5 to 3.2 x lox daltons (Reimer et al., 1966; Hoyle, 1968; Scholtissek et al., 1969) of which 7075% is protein (Frommhagen et al., 1959; Reimer et al., 1966). Secondly, the calculation was done using the current best estimates for the molecular weight of the influenza genome of 5.9 x lo6 (McGeoch et 1 OF FOWL

Percentage ion protein Stained

PLAGUE

VIRION

of total virby scanning* gel

Autoradiogram

POLYPEPTIDES

Estimated molecules Method

number of per vii-ion lc

Method

2d

1.1

0.7

15

14

2.2

1.4

34

32

21.6 18.1 6.1 16.6 34.3

26.9 23.1 4.3 15.4 28.2

1064

1000

1085

1018

211 1216 2468

198 1141 2315

a Molecular weights estimated by comigration on polyacrylamide gels using as markers: p-galactosidase, phosphorylase A, bovine serum albumin, immunoglobulin g (heavy chain), ovalbumin, lactate dehydrogenase, immunoglobulin g (light chain), &lactoglobulin, and cytochrome c. b Data obtained from 9% gel; P, and P, were not separated under these conditions. c Based on 2.1 x IO” daltons of protein per virion. rl Based on genome RNA molecular weight of 5.9 x lo6 representing 10% of RNP molecular weight.

494

INGLIS

al., 1976; Pons, 1976) in conjunction with the fact that purified influenza ribonucleoproteins contain between 10 and 12% RNA (Pons et al., 1969; Krug, 1971). Therefore the RNP contains about 53 x lo6 daltons of protein per complete genome and hence per virion, assuming one complete genome per virion. Since the molecular weight of the NP polypeptide is 53,000, there are approximately 1000 NP polypeptides per virion, and the numbers of other polypeptides per virion can be calculated from their molar ratios compared with the NP polypeptide. The results obtained using the two parameters are in good agreement (Table l), and confirm previously published estimates (White, 1974). The three P polypeptides are the least abundant virion polypeptides; this would support the notion that they are functional rather than structural polypeptides. Virus-Specific Polypeptide Infected Cells

Synthesis

in

ET

AL.

in Fig. 3, migrating in front of the M protein during electrophoresis of infected cell extracts. The M polypeptide appeared later in the infectious cycle than the NS polypeptide, as reported by Skehel (1973). Proof that the NS and M polypeptides are separate gene products was obtained by peptide mapping experiments. M and NS polypeptides from infected cells, as well as M polypeptide from purified virions, were labeled with l”S,lmethionine and separated by electrophoresis on SDS-polyacrylamide gels as before. The appropriate bands were cut out, digested with trypsin, and the resulting peptides were analyzed by a two-dimensional fingerprinting technique involving electrophoresis in the first dimension followed by ascending chromatography in the second. Figure 4a and b shows the peptide maps obtained from [““Slmethionine-labeled M protein from purified virions and from infected cells, respectively. The two maps are identical. The peptide map obtained from putative nonstructural protein from infected cells (Fig. 4c) is quite distinct from that of M protein, and we have found that this pattern is completely reproducible in separate experiments. We conclude that the virus M protein and the virusinduced NS protein are separate influenza gene products.

Monolayer cultures of CEF cells on 5-cm petri dishes were infected with fowl plague virus and the polypeptides were pulse-labeled for 15min periods at various times up to 4.5 hr postinfection (Fig. 3). Mockinfected control cultures were also analyzed. The structural polypeptides of the virus were identified in infected cells using proteins of purified fowl plague virus as Association of Three P Polypeptides with the Influenza Polymerase Complex markers. When polypeptides were pulselabeled late in the infectious cycle, seven The major polypeptide associated with polypeptides could be identified which influenza virus-induced RNA-dependent were not present in mock-infected cells. Of RNA polymerase activity in infected cells these, five comigrated with virion strucis NP (Schwarz and Scholtissek, 1973; tural polypeptides; these were P,, Pp, Px, Compans and Caliguiri, 1973). Indeed it NP, and M. Virion polypeptides HA,, and was postulated that the NP protein alone HA, and NA were not labeled in infected was the virus-induced RNA polymerase cells during a 15-min pulse of 13”Slme- (Compans and Caliguiri, 1973). Subsequently it was found that in addition to thionine. However, pulse-chase experiments confirmed that polypeptide HA is a NP, a high molecular weight polypeptide primary gene product and that cleavage of P (only one identified) was associated with the polymerase activity isolated from inthis molecule to polypeptides HA, and HA, is rapid and complete in CEF-infected with fected cells (Caliguiri and Compans, 1974). We have isolated a RNA-dependent fowl plague virus as previously reported by others (Klenk et al., 1972; Klenk and Rott, RNA polymerase active fraction from the 1973; Skehel, 1972). cytoplasm of fowl plague virus-infected cells by a procedure involving discontinThe influenza virus-induced nonstructural polypeptide (NS) can be clearly seen uous density gradient centrifugation fol-

INFLUENZA

VIRUS

GENE

495

PRODUCTS

NP

U

0.5

1.0 FPV-INFECTED

1.5

2.0 CELL

2.5

3.0

3.5

4.0

4.5

POLYPEPTIDES

FIG. 3. The synthesis of polypeptides in fowl plague virus-infected CEF cells. Infected and uninfected cells were labeled with [“YQmethionine for 15 min at various times after infection, immediately before harvesting. Whole cell lysates were subjected to polyacrylamide gel electrophoresis and processed for autoradiography as described in Materials and Methods. Equal amounts of cell protein were loaded onto each track. U, uninfected cell lysate; other tracks represent infected-cell lysates. The numbers indicate hours after infection. Unlabeled purified fowl plague virus was coelectrophoresed as a marker for virion polypeptides. Polypeptide separation is on a 15% slab gel and migration is from top to bottom.

lowed by rate zonal density gradient centrifugation (Compans and Caliguiri, 1973). The gradient was fractionated and each fraction was assayed for RNA polymerase activity (Fig. 5a). The polymerase complex sedimented just ahead of the larger ribosomal subunit. The peak fractions were pooled, precipitated with 0.5 N trichloroacetic acid, and the precipitate was electrophoresed on an 11% polyacrylamide slab gel; after drying, the gel was autoradi-

ographed and the X-ray film scanned at 450 nm (Fig. 5b). The only labeled polypeptides present were the NP polypeptide and polypeptides PI, Pp, and P, (Fig. 5b). Uninfected cells treated in an identical manner gave zero RNA polymerase activity in the final sucrose gradient and no labeled polypeptides were detected. The polypeptides associated with fowl plague virion polymerase were also examined. A number of techniques was applied

496

INGLIS

ET AL.

tryptic peptide maps of (a) M protein from [““Slmethionine-labeled purified FIG. 4. Two-dimensional plague virus, (b) M protein from l”“S]methionine-labeled infected cells, and (c) NS protein 13”S]methionine-labeled infected cells. Cells were labeled with [W]methionine (20 &i/ml) from 2-6 hr infection and processed for polyacrylamide gel electrophoresis. Protein-containing bands were excised slab gels, and tryptic peptide mans were prepared as described in Materials and Methods. Peptides applied to the spot marked 0. _

including disruption with nonionic detergents or sodium deoxycholate, or separation in a polyethylene glycol-dextran twophase system (Bishop and Roy, 1972; Hefti et al., 1975). Using the latter technique, 80% of the original polymerase activity was recovered from the dextran phase. The polypeptides associated with the dextran phase were P,, P,, P3, and NP, as well as small amounts of envelope polypeptides (Fig. 6). RNP purified either from whole virus and using sodium deoxycholate or from infected allantoic fluid by acid precipitation and CsCl density gradient centrifugation lacked the P polypeptides and had no detectable polymerase activity. Peptide Maps of P Polypeptides The unique nature of each of the three P polypeptides was confirmed by tryptic pep-

fowl from after from were

tide mapping. Since the P proteins constituted such a small fraction of total protein in the virion, we were unable to obtain sufficient radioactivity for mapping purposes from this source. Consequently, P polypeptides labeled in infected cells were used. [3”Slmethionine (200 $3/ml) was added to the cell culture medium from 2 to 6 hr after infection, following which the cells were processed and electrophoresed in a 7.5% polyacrylamide slab gel. Polypeptides P,, Pp, and P, were clearly separated by this procedure (Fig. 71, and were excised from the gel, digested with trypsin, and peptide maps of each were prepared as described earlier. Figure 8 shows the maps obtained, which although complex, are clearly different from each other. The maps were completely reproducible in separate experiments, and when mixtures

b

0 c FIG.

FIGURE

497

4b

4c

498

INGLIS

ET AL.

b

II

9

FRACTION

NUMBER

~$65

II

NP

HA,

II %

NS~ A

FIG. 5. Polypeptides associated with RNA polymerase complex isolated from fowl plague virus-infected cells. CEF cells were labeled with [‘~SS]methionine from 3-6 hr after infection, then disrupted and the polymerase activity isolated on a discontinuous sucrose gradient followed by a rate zonal sucrose gradient, as described in Materials and Methods. The gradient was fractionated and fractions were assayed for RNA polymerase activity (a). Fractions 5 and 6 were precipitated with TCA and the precipitates processed for electrophoresis. (b) Densitometer tracing of an autoradiogram of [35Slmethionine-labeled polypeptides separated on an 11% polyacrylamide slab gel. Purified unlabeled fowl plague virus was coelectrophoresed as a marker for virion polypeptides.

of P, and P, bands excised from gels were run together, the unique spots of each polypeptide were clearly identifiable. We conclude that there are three high molecular weight polypeptides associated with RNA polymerase activity of fowl plague virus, and that these are three separate gene products. DISCUSSION

The development of the SDS-trisglytine-buffered gel system of Laemmli (19701,based on the original work of Davis (19641, led to the identification of many previously unknown proteins of bacteriophage T4. Applying this technique to influenza virus-infected cells, our results indicate that eight distinct polypeptides with a total molecular weight of about 5 x lo5 are specified by the influenza (fowl plague) virus genome. Although this greatly exceeds the available coding capacity of the genome molecular weight as estimated by chemial analysis (Ada and Perry, 1954) or length measurements in the electron microscope (Li and Seto, 19711,recently studies of the influenza virus genome by poly-

acrylamide gel electrophoresis in presence of urea have led to a revised estimate of 5.9 x lo6 for the total molecular weight (McGeoch et al., 1976; Pons, 1976). The correlation between the eight pieces of influenza virus RNA and the eight polypeptides specified in infected cells is exceedingly good (Table 2). Moreover, in extensive studies with temperature-sensitive mutants of influenza virus, Hirst (1973) identified eight recombination groups, in agreement with the present data. The resolution of polypeptides M and NS on polyacrylamide gels has in the past proved difficult, and Gregoriades has cast doubts on their separate identity (Gregoriades, 1973; Gregoriades and Hirst, 1975). On the other hand, Lazarowitz et al. (1971) published one-dimensional tryptic peptide maps of the virion M protein and the nonstructural protein from infected cell nuclei which showed them to be different. Using slab gel electrophoresis, we were able to separate polypeptides M and NS and show that the two proteins are quite distinct, both from differences in their times of appearance following infection, and from

INFLUENZA

VIRUS

FIG. 6. Polypeptides associated with subviral RNA polymerase complex obtained by PEG-dextran phase separation. Fowl plague virus was disrupted with 1% NP40 and separated in a two-phase system of PEG-dextran as described by Bishop and Roy (1972). A portion of the dextran phase was precipitated with 4% TCA and the precipitate processed for electrophoresis as described in Materials and Methods. Densitometer tracing of Coomassie brilliant blue-stained 11% polyacrylamide gel. Unlabeled purified fowl plague virus was coelectrophoresed as a marker for virion polypeptides. Migration is from left to right.

two-dimensional tryptic peptide maps, confirming the conclusion reached by Lazarowitz et al. (1971). In addition to the 23,000-dalton nonstructural polypeptide considered here, a small polypeptide of molecular weight 11,000 has been found in infected cells and described as a second nonstructural polypeptide (Skehel, 1972; Krug and Etkind, 1973). We observed a polypeptide of about 11,000 daltons during analysis of infected cell extracts on 15 or 17.5% polyacrylamide gels, but not consistently. We have also occasionally detected a polypeptide with similar electrophoretic mobility in purified

GENE

PRODUCTS

499

including bromelainfowl plague virus, treated core particles (Carroll, 1976). Further investigation will be necessary to ascertain the nature of this 11,000 molecular weight polypeptide; however, no separate RNA species of a corresponding size was detected in the virion (McGeoch et al., 1976; Pons, 1976). The existence of three P proteins in influenza virus is supported by the following evidence: (i) Three P proteins were consistently detected both in purified virus and in pulse-labeled infected cells. (ii) The three P proteins were present in equal amounts. Where two P proteins have been reported previously, they were present in a ratio of 1:2 (Skehel and Schild, 1971; Skehel, 1972). (iii) The three largest RNA segments found in the influenza virion have sufficient coding capacity for three polypeptides of molecular weight about 90,000 (McGeoch et al., 1976; Pons, 1976). (iv) Two-dimensional tryptic peptide maps of P polypeptides from infected cells showed P,, PZ, and P, to be quite distinct.

FIG. 7. Polyacrylamide gel electrophoresis of [Yllmethionine-labeled high molecular weight polypeptides synthesized in fowl plague virus-infected CEF cells. Densitometer tracing of an autoradiogram. Cells were labeled with [Y3lmethionine (200 &i/ml) from 2-6 hr after infection. The cells were then harvested and prepared for electrophoresis as described in Materials and Methods. Purified unlabeled fowl plague virus was coelectrophoresed as a marker for virion polypeptides. Separation is on a 7.5% gel, and migration is from left to right.

500

INGLIS

*

ET

AL.

I ELECTROPHORESIS

’

L

FIG. 8. Two-dimensional tryptic peptide maps of (a) P,, (b) P,, and (c) Pi proteins obtained from infected cells. Bands containing the appropriate proteins were excised from the slab gel shown in Fig. 8 and tryptic peptide maps prepared as described in Materials and Methods, except that electrophoresis was carried out for 4 hr.

All three P polypeptides appear to be necessary for RNA polymerases purified either from influenza virions or influenza virus-infected cells. This would put the total size of the influenza RNA polymerase as 268,000 daltons; the active enzyme complex contains the RNP in addition. Other negative strand viruses appear to possess RNA polymerases of similar size. For example, in vesicular stomatitis virus the polymerase consists of the RNP plus a 230,000-dalton enzyme containing the L and NS polypeptides (Emerson and Yu, 1975). Similarly, a polymerase isolated from Sendai virus-infected cells consists of the RNP plus a 240,000-dalton enzyme containing the HMW and P polypeptides (Lamb, 1974; Lamb et al., 1976). The exact roles played by the various polypeptide subunits are not known for any of these

viral polymerases (Bishop and Flamand, 1975). Although the results described here were all obtained using the fowl plague strain of influenza A virus, similar studies have been carried out in our laboratory with purified egg-grown preparations of influenza A strains BEL and NWS. These strains have H,-type hemagglutinin and N,-type neuraminidase as distinct from Hav, and Neq, of fowl plague virus. Both strains contained polypeptides which comigrated during slab gel electrophoresis with the P, NP, and M polypeptides of fowl plague virus. In particular, three P polypeptides were observed in both these strains when analyzed on 7% polyacrylamide gels. Therefore, it seems likely that our findings represent a general feature of the polypeptides of influenza A viruses.

El.ECtf?OPHORESlS FIG.

8b

ELECTROPHORESIS

FIGURE SC 501

0

I c

502

INGLIS

ET

TABLE RNA RNA

Genome Molecular

weight

AND

PROTEINS

2

OF FOWL

PLAGUE

RNA”

VIRUS

Virus

Coding capacity (mol. wt.)

proteins

Molecular weight

Name

1 2 3

1.19 x 106 1.02 x 106 1.00 x 106

117,000 100,000 98,000

96,000 87,000 85,000

P, P, P,

4 5 6

0.83 x 106 0.68 x lo6 0.58 x lo6

82,000 67,000 57,000

75,000 53,000 45,000

Hemagglutinin Nucleocapsid Neuraminidase

HA NP NA

7 8

0.32 x lo6 0.28 x lo6

31,000 27.000

25,000 23.000

Matrix M Nonstructural

NS

a Data

obtained

in this

laboratory,

based

on McGeoch

ACKNOWLEDGMENTS We thank R. D. Barry and D. McGeoch for helpful discussion and criticism. This work was supported by grants from the Medical Research Council. S.C.I. and R.A.L. were recipients of MRC scholarships for training in research methods; A.R.C. was the recipient of a CASE scholarship funded by the Science Research Council and Fisons Ltd., Loughborough, England. REFERENCES G. L., and PERRY, B. T. (1954). The nucleic acid content of influenza virus. Amt. J. Exp. Biol. Med. Sci. 32, 453-468. BISHOP, D. H. L., and FLAMAND, A. (1975). Transcription processes of animal RNA viruses. In “Control Processes in Virus Multiplication” (D. C. Burke and W. C. Russell, eds.), pp. 95-152. Society for General Microbiology Symposium, 25. Cambridge University Press, London. BISHOP, D. H. L., and ROY, P. (1972). Dissociation of vesicular stomatitis virus and relation of the virion proteins to the transcriptase. J. Viral. 10,234243. 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. BRAND, C. M., and SKEHEL, J. J. (1972). Crystalline antigen from the influenza virus envelope. Nature New Biol. 238, 145-147. CALIGUIRI, L. A., and COMPANS, R. W. (1974). Analysis of the in vitro product of an RNA-dependent RNA polymerase isolated from influenza virusinfected cells. J. Viral. 14, 191-197. CARROLL, A. R. (1976). Influenza virus RNA transcriptase. Ph.D. Thesis, University of Cambridge. CARROLL, A. R., MCGEOCH, D., and MAHY, B. W. J. ADA,

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GENE

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