The polypeptides of influenza virus

VIROLOGY

49,

758-765 (1972)

The Polypeptides

of Influenza

Virus

VI. Composition of the Neuraminidase’ IEVA LAZDINS, School of Microbiology,

ELIZABETH University

A. HASLAM,

of Melbourne,

Parkville

AND

D. 0. WHITE

3062, Victoria,

Australia

Accepted June Ii, 1972

The neuraminidase of influenza B/Lee is a polymer composed of 4 molecules of glycoprotein of M, 68,CKlQ.Disulfide bonds link the monomers into pairs which in turn aggregate by noncovalent bonds. A second protein of M, 56,900 is found in smaller amounts in some preparations. Neuraminidase released from the virion by trypsin treatment differs from that released by sodium dodecyl sulfate (SDS) in that it cannot aggregate, migrates somewhat differently in electrophoresis, has a slightly lower molecular weight, and contains relatively less glucosamine. On electrophoretic analysis the monomer from the trypsin-derived enzyme is found to be exclusively of 56,060 M,. The evidencesuggests that it is derived from the “natural” glycoprotein by the loss of a carbohydrate-rich fragment from the hydrophobic (envelope-associated) region of the molecule. INTRODUCTION

The neuraminidase of certain strains of influenza may be released in active form from the virion by treatment with sodium dodecyl sulfate (Laver, 1963). The enzyme thus obtained has a sedimentation coefficient of 8-10s (M, 150,000-300,000). A M, of 150,000has been calculated from direct measurements of the dimensions of the enzyme in the electron microscope (Laver and Valentine, 1969), or 240,000 on polyacrylamide gels (Kilbourne et al., 1972). When analysed by acrylamide-gel electrophoresis after denaturation by heating in the presence of agents reducing disulfide bonds, the enzyme dissociates into glycoprotein monomers of iM, about 60,000 (Haslam et al., 1970b: Webster, 1970; Skehel and Schild, 1971; Kilbourne et al., 1972). There is some confusion, however, on the question of whether the enzyme contains one or more than one species of glycoprotein. Webster (1970) and Kilbourne et al., (1972) found two closely spaced electrophoretic bands, while Skehel 1 Supported by the Australian Committee.

Research Grants

and Schild (1971) reported two with one strain of virus and one with another, and more recently Laver (personal communication) and Skehel (personal communication) both found only a single band. In this paper an attempt is made to explore the reasons for the differences by comparing neuraminidase derived from the virus by treatment with trypsin (No11et al., 1962) and SDS (Laver, 1963), respectively. Evidence is presented that the smaller of the proteins may be an artifact, possibly derived from the larger by proteolytic cleavage, and that the active neuraminidase is, in fact, a polymer composed of a single speciesof glycoprotein. MATERIALS

Most of the materials and methods used in this work have already been described in detail in Taylor et al. (1969) and Haslam et al. (1969,1970a, b). Virus and cells. Stocks of the B/Lee strain of influenza virus were maintained by growth in fertile hen’s eggs. Radioactively labeled virus from eggs was obtained by injecting [Wjmixed amino acids into eggs 24 hr after infection and incubating the eggs for a further 48 hr (Haslam

758 C opyright All righta

0 1972 by Academic Press, of reproduction in any form

Inc. reserved.

AND METHODS

COMPOSITION

OF INFLUENZA

et al., 1970a). Alternatively radioactively labeled virus was obtained by propagating Lee for 3-4 days in primary embryonic calf kidney monolayer cultures (Taylor et al., 1969) in the presence of [W]mixed amino acids, or [aH]valine, or [BH] valine and [W]glucosamine. Purification of virus was by differential centrifugation, followed by rate sonal sedimentation in 15-60% (w/v) sucrose gradients as described by Haslam et at. (1970a). All radioactive virus grown in calf kidney cells was supplemented with unlabeled egg-grown virus in experiments requiring assay of enryme activity. No difference in electrophoretic mobility was discernible between the neuraminidaaes from the two host cell systems. Isolation of neuraminidase by SDS treatment. Sodium dodecyl sulfate (SDS) was added to the virus suspension to a final concentration of 2% and left at room temperature for 15 min. The mixture was then layered onto a gradient of 5-20% (w/v) sucrose and centrifuged at 81,000g for 17-18 hr at 20 C. The gradient was harvested aa 20 fractions and assayed for neuraminidase. The fractions of highest neuraminidase activity were pooled and dialysed overnight against phosphate-buffered saline, pH 7.2, at 4 C to remove sucrose and SDS. Volumes were reduced by vacuum dialysis. Isolation of neuraminiduse by trypsin treatment. Virus ww incubated with 5 mg/ml trypsin for 15 min at 37 C and centrifuged at 100,000g for 1 hr at 4 C. The supernatant fraction was then layered onto a 5-20yo sucrose gradient and centrifuged aa above. Dissociation of virus or neuraminidase for electrophoresis. Samples were prepared for electrophoresis by the following three methods. (1) Nonreducing conditions: SDS was added to the sample to a final concentration of 2% and allowed to stand at room temperature for 15 min. Samples were then dialysed against 0.1% SDS in 0.01 M sodium phosphate buffer pH 7.2 at room temperature for at least 4 hr. Samples were subjected to electrophoresis for 19.2 hr at 50 V on 5% polyacrylamide gels containing 0.1% SDS and 0.5 M urea, at pH 7.2. (2) Reducing conditions: Samples were SUBpendedin2% SDS, 0.5 M urea, 0.04 M 2-mercaptoethanol, and 0.1 M 2-hydroxyethyldisulfide in 0.01 M sodium phosphate buffer, pH 7.2, and incubated at 100 C for 1 min. Dialysis was against 0.1% SDS, 0.5 M urea, pH 7.2. Electrophoretic conditions were aa above. (3) Reducing conditions during dissociation and electrophoresis: Samples were suspended in 2% SDS, 1% 2-mercaptoethanol in 0.01 M sodium phosphate buffer, pH 7.2, and heated at 80 C for 30 min. Dialysis was against 0.1% SDS, 1%

VIRUS NEURAMINIDASE

759

2-mercaptoethanol, 8 M urea in 0.01 M sodium phosphate buffer. These samples were analysed by electrophoresis on 5’% polyacrylamide gels soaked for 2 days in 0.1 M sodium phosphate buffer containing 0.1% SDS and 1% 2-mercaptoethanol. Estimation of molecular weights. The following protein markers were used to estimate molecular weights of virus proteins: human y globulin, bovine serum albumin, trypsin, soybean trypsin inhibitor, and cytochrome c. Recovery of neuraminidase activity from gels. After electrophoresis, the gels were ground, collecting fractions into 1.5~ml volumes of 0.01 M sodium phosphate buffer, pH 7.2. Fractions were allowed to stand for a few hours at 4 C to facilitate the elution of protein from the macerated gel. The supernatant fluids of all fractions were assayed for neuraminidase activity by the method described in Haslam et al. (1970a). Radioactive labeling of neuraminidase by iodination with l*6Z. Highly purified neuraminidase was dissociated by treatment with 2% SDS at room temperature and dialysed against 0.1% SDS and 0.01 M phosphate buffer (pH 7.2) overnight. A 50-~1sample of dissociated neuraminidase (10-15 pg protein) was iodinated as described in Stanley and Haslam (1971). Briefly, the sample was incubated ,for 15 min at room temperature with 2 ~1 lrrsI (1 mCi/ml in 5.5 X lO+ M HI) plus 10 ~1 chloramine T (0.5 mg/ml in phosphatebuffered saline (PBS)). The reaction was stopped by addition of 10 ~1 potassium metabisulfite (1.25 mg/ml in PBS) and 0.2 ml PBS. Unreacted iodide was removed by dialysis against 0.1% SDS in 0.01 M phosphate. RESULTS

Neuraminidase Aggregation tion by Trypsin

and its Aboli-

To investigate the differences between the neuraminidase molecule released by SDS (SN) and that liberated by trypsin (TN) the sedimentation behaviour, electrophoretic mobilities, and glucosamine content of the two enzymes were compared. When TN and SN were centrifuged on parallel gradients of 5-20% sucrose both sedimented at the same rate. However, when subjected to a second sedimentation it was found that, whereas TN sedimented once again at the same rate as in the first gradient, SN was now heterogeneous and moved considerably faster (Fig. la and b) presumably as a result of the aggregation demonstrated electron microscopically by Laver and Valentine

760

LAZDINS,

IIASLAM,

ANI> WHITE Recovery of Active Acrylamide Gels

Seuramitridase

jrwla

Further differences betJveen SN and TN were revealed by acrylamide gel electrophoresis; if not subjected to any denaturing process,both types of purified enzyme could be recovered in active form from fractions near the top of the gels. However, TN was found to peak in fraction 7 regardless of whether the gel and electrophoresis buffer contained SDS, whereas the peak of SN was recovered from fraction 13 when subjected to electrophoresis in the presence of SDS and from fraction 7 when SDS was omitted (Fig. 2). Neuraminidase could also be recovered in active form after electrophoresis of whole virus and hence the relationship of the peak of enzyme activity to t,he other viral proteins on gels was established. [‘“Cl mixed amino acid-labeled Lee grown in eggs was disrupted with 2% SDS at room temperature, and subjected to polyacrylamide gel electrophoresis. The neuraminidase act’ivity in fraction 13 (about 12 % of the original

Fm. 1. Sedimentation in a second sucrose gradient of Lee neuraminidase released from the virion by trypsin or by SDS treatment. (a) Trypsin-derived neuraminidase was harvested from a sucrose gradient, dialysed overnight against phosphate-buffered saline, pH 7.2, and reduced in volume by vacuum dialysis. The enzyme was then layered onto a 540% (w/v) sucrose gradient. (b) SDS-derived neuraminidase harvested from a sucrose gradient was dialysed to remove the sucrose and SDS, reduced in volume as above, and layered onto a similar sucrose gradient. (c) SDS-derived neuraminidase was obtained and treated as (b) except that prior to layering onto the sucrose gradient it was incubated with 0.5 mg/ml trypsin for 15 min at 37 C. The three gradients were centrifuged in parallel at 81,OOOgfor 17 hr at 20 C and analysed for neuraminidase activity.

(1969). Moreover, if SN was treated with trypsin before the second sedimentation its aggregating ability was destroyed and the activity now sedimented at the same rate as TN (Fig. 1~).

__ FRACTlDN

NUMSER

FIG. 2. Electrophoresis of trypsin-derived and SDS-derived neuraminidases in the presence or absence of SDS. Concentrated samples of egggrown Lee were treated with either trypsin or SDS to release the neuraminidase which was then isolated on a sucrose gradient. The purified enzymes were then subjected to electrophoresis on parallel gels. The gel fractions were assayed for neuraminidase activity. Migration in this and all subsequent electrophoretograms is toward the anode on the right.

COMPOSITION

OF INFLUENZA

neursminidase activity of the virion) corresponded to a minor peak of radioactivity in the gel of whole virus (N, Fig. 3). This peak was estimated by the method of Shapiro et al. (1967) to have a iWr of about 150,000-250,000. The other electrophoretic components of the virion evident in Fig. 3 are unreduced haemagglutin (HAU), ribonucleoprotein (RNP) and internal (or membrane) protein (IP or RIP) corresponding to VPl, VP2, and VP3, respectively, of Haslam et al. (1970b). The minor peaks designated N, NZL, and Ns , are shown in the present study to be associated with the neuraminidase in different states of aggregation. This was demonstrated by examining the electrophoretic patterns of the neuraminidase under increasingly more stringent dissociation conditions.

VIRUS

NEURAMINIDASE

761

I

Substructure of the Neuruminidase

Enzyme from Lee virus grown in the presence of [14C]mixed amino acids was liberated by SDS treatment and purified on

r

Fra. 3. Acrylamide gel eleotrophoresis of influenza virus B/Lee after dissociation with SDS; location of active enzyme on the gel. Lee grown in eggs in the presence of [‘4C]mixed amino acids was dissociated with 2% SDS and subjected to gel electrophoresis. The gel was fractionated and allowed to soak overnight. The supernatant fluid of each fraction was assayed for neuraminidase and the remainder counted in a scintillation spectrometer (Haslam et al., 1970a).

FIG. 4. Comparison of the electrophoretic mobilities of the proteins of neuraminidase before and after reduction. Pure Lee grown in calf kidney cells in the presence of [Wlmixed amino acids and supplemented with unlabeled concentrated Lee from eggs was used to isolate neuraminidase. The enzyme preparation was then mixed with [JH]valine-labeled Lee virus from calf kidney cells. One half of the mixture of neuraminidase and virus was treated with 2% SDS, and analysed by gel electrophoresis (Fig. 4a). The other half was treated with 2% SDS, 0.5 M urea, 2-mercaptoethanol, and 2-hydroxyethyldisulfide (Fig. 4b).

a sucrose gradient. Half the preparation was analysed by electrophoresis without further treatment other than 2% SDS, in the presence of SDS-dissociated [3H]valinelabeled marker virus, while the other half was further denatured by heating in the presenceof 2 % SDS, 2-mercaptoethanol and 2-hydroxyethyldisulfide before electrophoresis in the presence of a similarly treated marker. As shown in Fig. 4a the unreduced enzyme was resolved into two main components, N,, and Ns , which correspond with minor components in the marker virus. There was no evidence of contamination of

762

LAZDINS,

HASLAM

AND WHITE

the purified neuraminidase by detectable amounts of the major viral proteins HAU, RNP, or IP. On reduction of the enzyme (Fig. 4b) the amount of radioactivity in the minor component N8 remained unchanged, but NzI, was converted quantitatively into a more rapidly migrating component NL . The increase in radioactivity in the RNP region of the reduced marker virus and the extra peak migrating to the left of the internal protein are caused by partial breakdown of the haemagglutinin peak, HAU, under reducing conditions to its two constituent polypeptide chains (Haslam et al., 1970b; Stanley and Haslam, 1971). Thus, NL migrated in the region of the nucleocapsid and the large chain of the haemagglutinin which were not resolved as separate peaks. The molecular weights of Ns , NL , and NZL were calculated, by the method of Shapiro et al. (1967), to be 56,000, 63,000, and about 110,000, respectively, suggesting that NZL is a dimer of N, . Detection of Carbohydrate in the Neuraminidase

To determine whether neuraminidase is a glycoprotein, the enzyme was purified from Lee virus which had been labeled by the incorporation of [14C]glucosamineas well as [3H]valine. Neuraminidase was isolated from one half of the virus preparation by SDS treatment and from the other half by trypsin. The purified SDS-derived enzyme was dissociated by 2% SDS, 2-mercaptoethanol, and 2-hydroxyethyldisulfide incubating for 30 min at 37 C only, to achieve partial reduction of NZL to NL (Fig. 5). Only NZL and NL incorporated glucosamine, both having a 14C: 3H ratio of approximately 0.06. This provides further evidence that NZL is a dimer of NL . The small component, N, , incorporated little or no glucosamine. In striking contrast, neuraminidase isolated from the virion by trypsin yielded only a single electrophoretic peak with a mobility similar to that of the small protein NB . Its 14C:3H ratio was approximately 0.03 which is half that (0.06) for the major component of SDS-derived neuraminidase. Closely similar ratios (0.04 and 0.07, respectively) were obtained in a second ex-

FIG. 5. Glycoproteins of Lee neursminidase. Radioactive Lee, labeled with [aH]valine and [“Clglucosamine and purified from calf kidney cells, was supplemented with nonradioactive concentrated Lee from eggs. Neuraminidase was extracted from half the virus preparation with SDS (a) and the other half with trypsin (b), then separated on a sucrose gradient, and prepared for electrophoresis by incubating with 2% SDS, 0.04 M 2-mercaptoethanol, 0.1 M 2-hydroxyethyldisulfide for 30 min at 37 C.

periment, suggesting that a glucosaminerich fragment is lost in the derivation of TN from SN. Analysis of Highly PuriJied Iodinated Neuraminidase

The question arose whether the small amounts of Ns associated with NL represented a contaminant or a degradation production of NL . Attempts to prepare more highly purified neuraminidase from virus labeled during growth in eggs or calf kidney cultures led to such losses of radioactive enzyme that alternatives had to be sought. Accordingly, unlabeled Lee was grown in eggs, purified, disrupted with 2 % SDS, and the neuraminidase isolated by rate zonal centrifugation on a sucrose gradient, then

COMPOSITION

OF INFLUENZA

further purified by acrylamide gel electrophoresis (after further treatment with 2% SDS). Gel fractions of peak enzyme activity were pooled, reduced in volume, and iodinated, in the presence of SDS, with 125I using chloramine T as oxidant. The radioactive enzyme preparation was halved and 2% SDS added to one half while the other was rigorously denatured and reduced by heating in the presence of SDS and 2mercaptoethanol (method 3). The results are given in Fig. 6. The position of the peaks of a marker virus run in parallel are shown.

,

FIG. 6. Electrophoresis of highly purified W-labeled neuraminidase. Highly purified neuraminidase was obtained from egg-grown Lee virus by SDS disruption followed by rate zonal centrifugation to isolate the enzyme, then acrylamide gel electrophoresis in the presence of SDS. Active enzyme was recovered from the gel and labeled with rasI. Half of this labeled enzyme preparation (a) was analysed by gel electrophoresis after treatment with 2% SDS only, while the other half (b) was reduced with 1% 2-mercaptoethanol and 2% SDS at 36 C for 30 min. The gels were presoaked in (a) buffer or (b) 1% 2-mercaptoethanol in buffer, respectively.

VIRUS

NEURAMINIDASE

763

It will be noted that all proteins migrate somewhat more rapidly in Fig. 6 than in Fig. 4 because the presoaked gels used in this experiment have swollen. As in Fig. 4 it will be seen that the major constituent, Nz, , of the unreduced neuraminidase is converted on reduction to the monomer, NL . However, in this experiment the smaller protein, Ns is absent. DISCUSSION

The use of proteolytic enzymes to isolate neuraminidase usually yields an enzyme very similar in sedimentation coefficient (8.0-10.5 S) to that released from the virion by detergents (No11 el al., 1962; Seto et al., 1966; Laver and Valentine, 1969; Webster and Darlington, 1969; Rott et al., 1970). A closer comparison in the present paper of the two types of neuraminidase reveals t’hat the trypsin-derived enzyme (TN) differs from the SDS-derived enzyme (SN) in the following respects: (i) It cannot aggregate, its sedimentation in gradients being uninfluenced by SDS. (ii) The electrophoretic mobility of the active enzyme is not increased by SDS, even though the constituent monomers bind substantial amounts of SDS after complete reduction and denaturation. This suggests that the intact neuraminidase molecule contains a trypsin-sensitive hydrophobic region which preferentially binds SDS (Reynolds and Tanford, 1970). (iii) After reduction it migrates in gel electrophoresis as a single component of slightly lower molecular weight than NL and the same molecular weight as the protein Ns found in small amounts in some preparations of neuraminidase. (iv) Comparison of its [14C]glucosamine to [3H]valine ratio with that of the SDSderived enzyme suggests that a small peptide rich in glucosamine has been cleaved off each NL molecule. The data suggest that trypsin specifically cleaves off a small hydrophobic portion of the neuraminidase molecule that is not involved in the active site, but is responsible for aggregation, and is rich in carbohydrate. This is probably the “foot of the mushroom” by means of which SDS-derived neuraminidase molecules aggregate when

764

LAZDINS,

HASLAM,

SDS is removed (Laver and Valentine, 1969). The resistance of the neuraminidase of influenza B/Lee to SDS (Laver, 1963) permitted clear separation of the enzyme by rate zonal centrifugation. The haemagglutinin, ribonucleoprotein, and internal protein were all denatured and remained at the top of the gradient, whereas the enzyme sedimented as a single band which on subsequent electrophoretic analysis was demonstrably free of contamination by any of the three major viral proteins. In this respect Lee proved to be a more fortunate choice than A2, the haemagglutinin of which is partially resistant to SDS, and hence cannot be completely separated from the enzyme on sucrose gradients (Rott et al., 1970). Similar problems hindered Webster and Darlington (1969) in separating the neuraminidase from the haemagglutinin of Tween 20disrupted X-7 on sucrose gradients or Sephadex columns. In the case of Lee, however, the haemagglutinin was completely denatured by SDS while the neuraminidase survived. One result of this fact is that when SDS-disrupted Lee is analysed by electrophoresis on SDS-containing gels the high molecular weight polymers remaining near the origin are components of the neuraminidase in various states of aggregation, whereas with Bel, which has anSDS-resistant haemagglutinin and SDS-susceptible neuraminidase (Laver, 1964), they are aggregates of the haemagglutinin components (Stanley and Haslam, 1971). Although the neuraminidase preparations used in this study were shown to be free of contamination by the major viral prot’eins, the finding of the small protein, N, in various preparations is puzzling. This polypeptide is a very minor protein of unknown function seen in all preparations of purified Lee virus. The absolute amount of N, found in preparations of SN varies somewhat from experiment to experiment but it is always a relatively minor component, whether measured by radioactivity or by staining with Coomassie blue, as has also been the experience of Webster (1970). It was originally believed to be a neuraminidase component because it was found to co-

AND

WHITE

sediment with the 9 S molecule while the major viral prot’eins (HA, RNP, and IP) did exnot. However, when neuraminidase tracted with SDS from egg-grown Lee virus was separated on a sucrose gradient, then further purified by acrylamide gel electrophoresis and labeled with lz51, N, was no longer present. It is unlikely that iSs simply eluded detection by failure to be iodinated. Rather one must conclude that, since the electrophoretic purification step removed it leaving the enzyme still active, it is not a vital component of the neuraminidase molecule. It is possible that Ns is a proteolytic cleavage product derived from NL , either in vivo or during the protracted purification protocol. Cleavage of the adenovirus hrxon polypeptide into smaller polypeptides during storage and purification has recently been described (Pereira and Skehel, 1971). This hypothesis gains some support from the fact that trypsin-derived neuraminidase consists entirely of a polypeptide with the same electrophoretic mobility as Ns . However, it is by no means certain that Ns is identical with this trypsin derivative of N, . Peptide mapping would clarify the issue. The major neuraminidase glycoprotein NL has a IM, of about 63,000, i.e., approximately half that estimated for NzL (lOO,OOO120,000 in several experiments) and one quarter to one third that of the active enzyme N. (It must be borne in mind, however, that the straight-line relationship of migration to log molecular weight cannot be accepted with confidence to provide a really reliable answer for large glycoprotein polymers.) Stringent reduction with disulfide bond-splitting agents is necessary to convert Nz, to N, . In the presence of SDS alone the active enzyme, N, occurs as an equilibrium mixture with Nz, , the relative proportions of the two depending on such factors as the protein:SDS ratio and whether or not the preparat,ion is heated to 100 C. The evidence suggests, therefore, that pairs of NL molecules occur as disulfide-linked dimers, which in turn associate by noncovalent bonds to form higher polymers, probably tetramers. Webster (1970), Skehel and Schild (1971) and Kilbourne et al., (1972) have all found

COMPOSITION

OF INFLUENZA

that strong reducing conditions are necessary to dissociate neuraminidase irreversibly into the 60,000 M, monomers. The occurrence of two closely spaced bands with some strains yet only one with others, suggests that two distinct species of glycoprotein may be involved but that electrophoresis may not resolve them if they happen to be of identical molecular weight. If this is so, the structure of neuraminidase shows a striking resemblance to that of hemagglutinin (Skehel and Schild, 1971; Stanley and Haslam, 1971).

VIRUS NEURAMINIDASE

765

Morphology of the isolated haemagglutinin and neuraminidase subunits of influenza virus. Virology 38,105-119. NOLL, H., AOYAGI, T., and ORLANDO, J. (1962). The structural relationship of sialidase to the influenza virus surface. Vic7iroZogy18, 154-157. PEREIRA, H. G., and SKEHEL, J. J. (1971). Spontaneous and tryptic degradation of virus particles and structural components of adenoviruses. J. Gen. Viral. 12, 13-24. REYNOLDS, J. A., and TANFORD, C. (1970). Binding of dodecyl sulfate to proteins at high binding ratios. Possible implications for the state of proteins in biological membranes. Proc. Nat. Acad. sci. U.S. 66, loos1007.

ROTT, R., DRZENIEK, R., and FRANK, H. (1970). On the structure of influenza viruses. In “The The authors thank Mr. I. Barlow, Miss C. Biology of Large RNA Viruses” (R. D. Barry Luck, and Miss L. Strawbridge for excellent and B. W. J. Mahy, eds.) pp. 75-85. Academic technical assistance during this work. Press, New York. SETO, J. T., DRZENIEK, R., and ROTT, R. (1966). REFERENCES Isolation of a low molecular weight neuraminiHASLAM, E. A., CHEYNF,, I. M., and WHITE, D. 0. dase from in5uenza virus. Biochim. Biophys. (1969). The structural proteins of Newcastle disActa 113,402404. ease virus. Virology 39, 118-129. SHAPIRO, A. L., VIRUELA, E., and MAIZEL, J. V. HASLAM, E. A., HAMPSON, A. W., EGAN, J. A., (1967). Molecular weight estimation of polypepand WHITE, D. 0. (1970a). The polypeptides of tide chains by electrophoresis in SDS-polyacrylinfluenza virus. II. Interpretation of polyacrylamide gels. Biochem. Biophys. Res. Commun. 26. amide gel electrophoresis patterns. Virotogy 42, 81.5820. 555-565. SKEHEL, J. J., and SCHILD, G. C. (1971). The polyHASLAM, E. A., H-4MPSON,A. W., RADISKEVICS, I., peptide composition of influenza A viruses. Virology 44, 396-40s. and WHITE, D. 0. (1970b). The polypeptides of influenza virus. III. Identification of the haeSTANLEY, P., and HASLAM, E. A. (1971). The polypeptides of influenza virus. V. Localization magglutinin, neuraminidase and nucleocapsid of polypeptides in the virion by iodination techproteins. Virology 42, 566-575. KILBOURNE, E. D., CHOPPIN, P. W., SCHULZE, niques. Virology 46, 764-773. I. T., SCHOLTISSEK, C., and BUCHER, D. L. TAYLOR, J. M., HAMPSON, A. W., and WHITE, (1972). Influenza virus polypeptides and antiD. 0. (1969). The polypeptides of in5uenza virus, I. Cytoplasmic synthesis and nuclear acgens-summary of influenza workshop I. J. Incumulation. Virology 39, 419-425. fee. Dis. 126, 447-455. LAVER, W. G. (1963). The structure of influenza WEBSTER, R. G. (1970). Estimation of the molecular weights of the polypeptide chains from the viruses. 3. Disruption of the virus particle and separation of neuraminidase activity. Virology isolated haemagglutinin and neuraminidsse subunits of influenza viruses. ViroZogy 49, 643-654 20, 251-262. LAVER, W. G. (1964). Structural studies on the WEBSTER, R. G., and DARLINGTON, R. W. (1969). Disruption of myxoviruses with Tween 20 and protein subunits from three strains of influenza isolation of biologically active haemagglutinin virus. J. Mol. Biol. 9, 109124. and neuraminidase subunits. J. Viral. 4,182-187. LAVER, W. G., and VALENTINE, R. C. (1969). ACKNOWLEDGMENTS