Structural investigations of influenza B virus

J. Nol. Biol. (1981) 151, 329-336

Structural Investigations of Influenza B Virus Purified preparations of influenza B/Hong Kong/5/72 have been characterized by hydrodynamical measurements, electron microscopy and small-angle neutron the preparations are highly scattering. As judged by these techniques monodisperse, the virus particles being spherical and of molecular weight about 200 x 106. The lipid bilayer is located at a radius of 425 A and its molecular weight is estimated to be 60 x 106, constituting about 3096 of the total virus mass. The

external radius is about 580 A. The ortho-myxo class of viruses contains the immunologically distinct influenza A, B and C: strains, all of which infect humans, the A strain also infecting ot’her animals. Influenza virus possesses a number of unique properties amongst which the antigenic variation related to its epidemological behaviour is well known. A recent collection of papers covering many aspects of influenza virus research is to be found by Schild (1979). The current’ low resolution picture of the virus structure is largely based on a combination of biochemical evidence and electron microscopy (Schulze, 1973 ; Laver, 1973; Wrigley, 1979; Skehel et al., 1980). There are four main structural prot,eins involved in the virus architecture. The outer envelope of the virus consists of a lipid bilayer from which two types of glycoprotein project externally. A minorit’y of these spikes consist of tetramers of neuraminidase, the majority being trimers of haemagglutinin on which reside the principal antigenic sites. The crystal st’ruct’ure of haemagglutinin has recently been solved (Wilson et al.. 1981) and a crystallographic investigation of neuraminidase is underway (Laver et al., 1980). Inside and supposedly in close contact with the lipid is thought to be a layer of socalled M-protein. This encloses a core of nucleoprotein which is largely composed of RXA complexed with the fourth main protein species. The RNA genome is segment,ed and of total molecular weight about 5 x 106, about 20/b of the virus mass. Even though an enormous amount of work has been done on structural aspects of influenza virus many uncertainties remain about both the chemical composition and the arrangement of the constituents. With the aim of providing a more secure structural basis for the understanding of t,he processes of infection and assembly we have begun a structural investigation of the virus using a number of physical techniques. Here we present preliminary results based on hydrodynamical measurements, electron microscopy and neutron small-angle scattering. The measurements were made on preparations of influenza B/Hong Kong/5/72

generously supplied by DUPHAR. Holland, and purified as described in the legend t’o Figure 1. A number of results obtained from sedimentation velocity and photon correlat,ion

experiments

are displayed

in Figure

1. Sedimentation

studies indicate

a

329 0022%2836/N/260329-08

$02.00/O

,C 1981 Academic

Press Inc.

(London)

Ltd.

1.0

2.0 [IV]

3.0

‘b

I

2 4 (IO5

(mg/ml) (a)

3

cm-‘)

(b)

2ot

1

I-

I

180

210

240

(x10’

(c)

-.I.> 270

daltons)

Cd)

FIG:. I, (a) A graph ~~S~~I~TSIIS concrnt.rittion of infiuezlza virus (IV) in 0.01 wphosphate (pH 7.5) atltl 0.75 M-KU. For the calculation ofs,,,, values the partial specific volume was taken as 0.79 and assumed to he independent, of ionic strength. The starting material for the purification steps consisted of inact,ivated influenza H/Hong Kong/S/72 suspended in 0.1 mphosphatje buffer. which was generousl! supplied by I)I%‘HAR (Weesp. Holland). The virus was grown on eggs and inactivated b,v treatment with 0.01 oh p-propiolartone. In a typical purification experiment about 2 1 of t,he inactivated virus suspension (wncentration about 1 ma/ml) was purified and cwwentratrtl I)y centrifugation in a Beckman continuous flow rotor on a 359, t,o 6%~ (w/v) linear sucrose gradient in PK buffer (05 wK(‘l. 0.01 M-phosphat)e buffer, pH 7.5). The fractions between 32 and 44’+, VW-c pooled and the sucrose was removed by centjrifupation. The virus pellet wax rrwspended in a total of about 20 ml PK buffer. This solution was layered on a linear 2X0 t,o 509, (w/v) sucrose gradient in PK buffer and a ratezonal centrifugation was carried out in a Beckman Ti-15 rotor for 3.5 h at 2d.oO0 revw/min. The frart,ions bet,ween 33?,, and 379 o sucrose were collrct~ed and concentrated by pelleting and rexuspending in PK buffer. Protein concentrations were estimat,ed according to the met,hod of Lowry rf nl. (19.51) with commercially available bovine serum albumin as a reference. I)ry weight mrasurement~s indicat,ed that the Lowry method gives 73 f 3’$, of the true virus concentration. (b) The results of phot,on correlation analysis. A plot of dzo,w v~~su~s scat,tering angle (q) for purified influenza H/Hong Kong in 001 MSa2HP0,, 0.75 M-KC] (pH 7.0). On t,he right vertical axis the Stoke’s radius is plotted. Virus concentrations are 0. 0.02 mg/ml; 0, 0.13 mg/ml: and 0. 1.3 mg/ml. The measurements were carried out in the apparatus described by Zulauf & Eirke (1979) at 2O’C’. The linewidt,h analysis was based cm the expansion of the logarithm of the first&order correlation function in terms of mommt,s (Brown pt nl 197.5). For each camera setting some 7 spectra were taken and the mean linewidth pand the 2nd and 3rd moments p2jiz and psrJ were determined individually The error bars in the Figure reflect the

LETTERS

TO THE

EDITOR

331

single species wit,h a s:~,~ of 651 S +25 S (see the legend to Fig. l(a)). This value is independent of virus concent’ration in the range from 1 to 5 mg/ml and of the ionic strengt)h of t,he buffer (@Ol M-phosphate, pH 7.5) in the range from 0.15 to 0.75 MKU. Quasi-elastic light scattering (see Fig. l(b)) gives a value of (3.45f0.05) x lo-* cm2 s-l for t,he translational diffusion constant di,,, which is independent’ of scatt,ering angle and virus concentrat’ion. This value is in good agreement with data obtained from an influenza A st’rain (Kharitonenkow et al., 1978). The c~orresponding hydrodynamic (Stoke’s) radius is 620 A. The photon correlation curves are well fitted by a single exponential decay, strongly suggesting a high degree of monodispersity (see t,he legend to Fig. l(b)). In order t’o deduce the molecular weight of the virus using these values of s and d in the Svedberg relation, it is necessary to know the partial specific volume accurately. At present this quantity can only be estimated (see Table I). A value of 0.79 cm3 g-* for i; gives a molecular weight of 217 x lo6 which decreases to 190 x lo6 when fi is 0.76. In order to obtain further information on t,he shape of the influenza virus particles. wit#h eit’her uranyl acetate or speGmens negatively stained phosphotungstate were prepared for elect’ron microscopy. It was found that the preparation procedure with both stains tended t’o disrupt, a certain percentage of t’he particles as also report’ed by Nermut &r Frank (1971), resulting in the occurrence of abnormally shaped particles and giving the impression of pleomorphism. Vranyl aceMe at pH 4.2 appears to have a less damaging effect than phospht’ungst’at8e solut’ions at pH 6.5. An electron micrograph with a number of well preserved sphericaal particles is shown in Figure I(c). The particles have a diameter of 1140 + 40 LA in uranyl acetate and 1225 f 40 x in phosphotungstat,e. An independent estimate of the molecular weight of the virus has been made using scanning transmission electron microscopy. This method. originally proposed by Zeitler & Bahr (1962), takes full advantage of the image recording mechanism availahlc in scanning transmission electron microscopy (Engel. 1978). The procedure used here will be described in det,ail elsewhere (I’. .I. Andree, R. R8uigrok & .J .I. I’. van dcr Voort, manuscript in preparation) but is based on a comparison of the irrt’egrated image int,ensity of unst’ained and air-dried influenza virus with a standard (t’obacco mosaic virus) of known molecular mass. In Figure l(d) a hist,ogram of the results is shown indicating a value of (178122) x 106 for t,hr molec~ular n-eight’ of influenza virus. This is in fairly good agreement with other determinat’ions reported in this letter.

varianws of’ t,hr results from these groups of spectra. For example the 2nd and 3rd moments are. respectively. OIIO9+0~012 and 0~018+0+26 for the sample with concentration 1.0 mg/ml. These values indicate a highly monodisperse sample. (c) An electron micrograph of a field of well-preserved negat,irely stained influenza virus. The negative stain is uranyl acetate of pH 4.2: the average diameter of the spherical particles is 1140 i\. The preparation procedure was essentially as described by Cremers et nl. (1981) and the specimens were air-dried. (d) A histogram of the influenza virus mass. as measured by scanning transmission electron microscopy at 60 kV in the instrument described b?- Schepman et al. (1978). The method used consisted of comparing the mass of unstained and air-dried influenza virus (IV) part,icles with the known mass of tobacco mosaic virus particles, which were mounted on the same thin carbon foils as the influenza virus particles. An average value of (178&22) x IO6 daltons wasdetermined.

Protein t RKA Cholesterol Phospholipidf Carhohydrate$

componrnt 2.3 I 325 0,344 0507 2Qo

3+Kl 417 0.813 ofl91 3.74

Scattering length/mass (B/M x IO-l4 cm/d&on) in ‘H,O in ‘Hz0 43.0 71.7 11.5 12.8 466

Match-point (“‘0 ‘H,O)

(‘t, ‘H,O)

specific vulumc~ (cm3/s)

Match-point

Partial

Scattering length relative to ‘H,O/unit mass ((X6’ - ~sb~),/M x 1OF I4 cm/dalton)*

0.73 053 0.95 @97 063

Partial specific volume (cm3/g)

31.0

0.789

2,485

0.62 0.02 0.13 0.17 0%

1

31.7

0.785

2.518

0.64 wo2 0.12 0.16 0%

Mass fraction)) 2

34.8

0.76fi

2.651

@72 0.02 @OS6 0.114 0.06

3

LETTERS

TO THE

EDITOR

333

O-020 0 I/angstroms FIN:. 2. (a) Small angle neutron scattering curves of influenza B/Hong Kong obtained at the indicated ‘H,0/‘H20 ratios. The measurements were made on instrument Dll at the Institute Laue-Langevin, Grenoble. France using specimen-detector distances of 19.8 and 9.8 m. a wavelength of 4.9A and a collimation of 20 m (Ibel, 1976). The virus concentrations varied from about 80 to 106 mg/ml. Additional measurements of the central maximum were made with concentrations of about 2 mg/ml, a specimendetector distance of 19.8 m and a wavelength of 13 A. The exact ‘H,O content of the buffers (975 MKC]. 0.01 M-phosphate. pH 7.5) was determined by density measurements and converted to equivalent pure ‘H,O/‘H,O mixtures with the same coherent scattering length density to take account of the high salt present. After circular integration all scattering curves were corrected for background and detector response. and put on an absolute scale using the incoherent scattering of the H,O buffers (R. May, K. Ibel & .J. Haas, unpublished results). (b) (Inset) A plot of P(O) against *Hz0 content. The best straight line through the points gives a match-point of 31.6% ‘Hz0 for the virus. The molecular weight can be estimated from this plot as described in the text. The 2 extreme points in the plot represent measurements at 0 and 1060/o ‘H,O. (c) Calculated scattering curves (solid lines) based on spherical shell models with 3 shells and fitted to the experimental data in 216 and 41.4% *H,O, respectively, indicated by crosses and circles. In 21.6% *H,O the radii of the shells (weights of the scattering length densities in brackets after each radius) are 584 A (969), 447 A (-2.63) and 467 A (160) and for the 41.1% ‘H,O data the tits were obtained by a distribution of shells specified by 5&1 A ( -0.03). 442 A ( - 190) and 398 .k (044).

334

?J E

MEL L E iI1 A I%7 .1 /A.

(‘orrwttd neutron scattering wr\~33 to a rrsolut~iori of l/160 X ’ and obtained from influenza virus solut)ions at’ a number of different ’ H20/2H20 ratios arr shou-n in Figure Z(a). At *H,O cont’ent,s ahow W),, thr, lipid dorninates the scdtwing giving rise t,o fivt> clearly rwolveci subsidiary maxima. At) lowtar ’ HZ0 conttwts the protein scattering contjribution is more significant. ~~spwially near thrl lipid match-point of lP,,, and the curves show marked changes with cont,rast. The cwtjral maximum of the watt~ering cm-w’s (not shou-n) can tw used to find the radius of gyration of the particks itnd the extSrapolated zw-o angle watt~rring I(O) as a fuwtion of csontra,st. Thr radius of gyrat’ion only shows small variations uith contrast (up t,o So,,), its value at infinite cont,rast being estimated to he 455 A. This radius of gyration would be given by a, homogenrous sphwical scattjerer 01’ radius 590 ,4. A plot of l+(O) ner.s/Ls2H,0 content is~shown in Figure d(b) from which a in the lipid content of thtx matd-poiirt of 31W,, is dduwd. I)ue to the uwrrtairity virus the, data, summarized in ‘I’ablt~ 1 havtb Iwen ustd to cd~~ulatt~ thti partial spedic volumes. scattering length pvr unit mass and match-point of thtl virlls tot. lipid cwntrwts twtwwn 20 and 300,,. The obst~r\~c4 mate-h-point is preclic~ttd 1)~.ii lipid wntrnt of 2H0,, with a c~or~r~~sy)ontlirlp virus rnolcc~ular wight of 219 X 105 ‘I’liis agrees very n~tdl with thtb ti~dr~otl?-narnic,al rnt,itsIirClrierits wport~etl abo\-tb. hut is considerably lowvr than valurs hithrrt,o rtyorttd in thv litthratuw for inllutwzw .A strains ((‘omparis & (‘hoppin. 1975). Spheric-al shrll models wtxre fitted to thr scat’ttaring vurvtv using a Icast-scluarw algorithm lvith the aim of deriving a wnsistvnt model fbr thv sckwt,tering It~ngth density in tticb partic-le (sw for instanw (‘hauvirr d c/l.. l!I7X: Schrwitlrr fd frl.. f 978) ,411curvt’s cari htk extremrly wdl fittjrd 1)~ this prowdurt- using frorn t hrw to tivtk shdls (see Fig. 3(c) for rxanlples) alt,hough thrw apTwars to Iw a litde dtlitional arlplllar-drf)t~rrc~t,rlt smearing of the wattrring f’ur\.( 3 aho\~rbthat espwt~ctl from ttiv irI ttic \vawlerigtli sprrad of Wo. This wulti arise from ;t narrow size distributioil particles (a (:aussian distribution of similar parGlr3 I\-it,h stautlartl drviatiori alwrit l(?,, of the mean radius would awountj for this ot)serrat~ion). from deviatjions front spherical symmetry (e.g. due to tfistortjabilitjy or the arra,ngement of thth spikes) or from multiple wattwing. The latjter is cdimattd to Iw negligible except ptvhitps for, the data in lOW;, ‘H, 0. \Ye c~orrvlucit~therefore that t.hca neutron watjtwing stjrongly supports tjtw trydrodynarnit~al measuwmfwts in drmonstrating t,hat t hv influrnza virus preparations are highly homoprncwus and contain particles lvhich t (I a resolution of 1,450 :I - ’ are twwntially sphwical and itlrntic*al. To aroitl ha,viry t,o det~rrnmitw t’oo many unkno~\~n paramettars two constraints \vcre applied during the model Wing. A lipid hilayv thickness of 40 AAwas imposd (to ctt~trrmitlt~ this thic~krwss is \\-rll Iwyorrd the mrasuwd rwolution) together with ii spike Ivngth of 135 A$ (Wilson rt ~1.. 1081 ). The most unnmI)iguously drtrrmintd quantit)y from the model fit,ting is thtx position of thtb lipid hilayer whosr cwttvr is at a radius of 42.5+5 w. This gives au outer radius of about 580 :I for the particle in good agreement \vitjh t)hr radius of gyrat)ion. hut A”,, less t ban t,he hydrodynam iv radius. Al tbstirttatr of the molwlllar weight of tilt, lipid cari IF made using values of 35 A2 and 47 A2 for the artaas wcupicd I)y diolr~stc~rol and pfiospholipid rnolrw~lt~s. rrspwtively. (Lrvinc~ & N’ilkins. 197 1). Taking tlw mdar f’r.ajc*tionof cholestc~rol as 0% (Tiffa,ny k Blough, 1070) the molecular wright of thth lipid is 62 x 106. This will

LETTERS

TO THE

335

EDITOR

he a slight overestimate due to embedded protein but agrees well with the 280/, lipid A more detailed interpretation of the eontent deduced from the match-point. scattering curves, in which it is hoped t’o elucidate t,he structure interior to the bilayer. awaits the results of neutron measurements on spikeless particles (obtained 1,~ protJease action) which will be undertaken soon. Some elementary conclusions about the number and spacing of the spikes in the cbnvelope of influenza B virus can be drawn from t’he molecular weight and dimensions reported above. Recent estimates suggest’ that the total mass of the spikes is between 35 and 45?/,, of that of the virus (Cusack et al., unpublished results), somewhat’ higher than previously thought (Schulze, 1973). Each spike is of molecular weight about’ 220,000 and therefore a particle weight of 220 x IO6 c~orresponds to 350 t,o 450 spikes. In the absence of knowledge about t’he symmetr) of the arrangement of the spikes (whether icosahedral or possibly with no longrange order) we assume that they are uniformly packed in a pseudo-hexagonal fashion. The near separation of the spikes 11 at’ radius R is then related to the number of spikes ,I: by N = 8~/3*. R21D2. .4t the external radius of 580 A the separation would therefore be 104 to 118 8. The crystal structure of the haemagglutinin spikes show that the maximum diamet’er is about’ 80 a (Wilson rf al., 1981) and a similar value is estimated for t’he neuraminidase heads (Laver, 1973). so that the preceding calculation could suggest, that the spikes are not tightl) packed. The results reported in this let,ter demonstrate conclusively that, monodisperse preparations of influenza B can routinely be prepared. t,his being an important prerequisite for the successful use of scattering techniques in t’he determination of the &uct,ure of t’he virus. In demonst’rating this we are led t’o hypothesise that there is a well-defined and unique struct’ure for infective influenza viruses (of a given strain and given host) in contradiction to the commonly held view that’ inflwnza

virus

is particularly

variable

in size and

shape.

\Ve are irrdrhted to DCPHAR (Weesp, Holland) for their generous donation of the influenza virus used in these experiments. \Ve also t,hank Dr .J, J Skehel and Dr B. Jacrot, for invaluable discussions and Drs P. Timmins and R,. IEay for technical assistance with the neutron experiments. This work \vas supported hy grants from the Setherlands Foundation for Chemical Research (SON) and the Netherlands Orpanizat,ion for t)he Advanrement of Pure Research (X12’()). Dcpartmr~nt of Biochemistry ITnivvrsity of Leiden \Vassenaarsrweg 64 2333 AL Leiden. Holland European ~lolecular Outstat,ion. (:renoblr,

Biology Laboratog France

“r .\uthor to whom correspondencr should be addressed. $ I’rewnt address: Department of Molecular Biophysics, I‘.K.

,J. E. MELLEMA~, P. J. ANDREE P. C. J. KRYGSMAS, C. KROOIC R. W. H. Rrw~~s

S. CVSSCK. A. MILLERS AND M. ZIXAVF

Park Road. I’nivcrsity

of Oxford, Oxford.

336

.J. E. MELLE&lA

E7’ .-1L.

KEFEKENCES Blouph. H. A. 8: hlerlie, ,J. I’. (1970). l’iroloyy, 40, 68.7.-692. Brown. .I. C., Pusey. 1’. N. & Dietz. R. (1975). .J. Chm. Ph,ys. 62. 1136 1144, Chauvin. t’., Witz, .I. bz Jaorot. B. (1978). J. A’ol. Biol. 124. 641-651. Cornpans. K. N’, & (‘hoppin. 1’. W. (1975). ln Comprrhrnsinr lTiroloqy (Fraenkrl-(lomat. H & RTagner. R. Il.. eds), vol. 4, Plenum Press, New York. t’remcrs, A. F. hf., Oost,ergetel, (1. T., Srhilstra, )I. & Mrllema. .I. E. (1981). J. ,Vo/. Hid. 145. 54.5~~ 56 1 Engel, =1. (197X). i’ltramicrosr(~ioy, 3. 273281. 9, 630-643. Ibcl. K. (1976). J. ‘-lppl. C’rystallogr. .Jacrot, B. & Zacpai. G. (1981). Riopolymers. in thr press. Kharitonenkow. I. (i.. Siniatov. 11. S.. Grigorirv. V. B., Aretier. I. Il., Eskor. A. I’. &

Levine, I’. K. & Wilkins. Ril. H, F. (1971). N&we AVCU$ Biol. 230. 69-72. Lowry, 0. H., Rosehrough, N. .J.. Parr, A. L. & Randa,ll. K. .I. (1951). J Biol. Phpm. 193, 265-275. Nermut. bl. V. & Frank. H. (1971). J. (:vN. I’irol. 10. 37 51. Srhepman. A. M. H.. van der Voort, .I. A. I’.. Kramer, *J & Mellema. .I. E. (197X). 1 TItramiuoscopy.

3. 265-969.

Schild. (:. (‘. (1979). /n.fltrur/sn. Brit. MN/. B*ll. 35. I 91. Schneider, I)., Zulauf. M.. Schafer, R. & Franklin, I~.. Al. (1978). J. ,110l. Viol. 124. 97 II”. Schulze. I. T. (1973). .-Ldvan. l’irrtx RPS. 18, I-55. Skehel. .I. .J.. Hay. A. J. & Wat.erlield, M. D. (1980). III C’c,l/Numhm~/rs avd I’iral Enwlopr.\ (Blough, H. A. & Tiffany, .J. M.. eds), vol. 2. pp. 647 681, Academic Press. London Tiffany, J. hI. & Blough. H. A. (1970). Proc. Snt. =I&. Sci.. I’.A’.;l. 65. 1105-111~. Ward, C‘. W. 8: Dopheide. ‘1’. A. (1979). Hri/. Med. J. 3.5. 51 56. Wilson. I. A., Skrhel, J. ,J. & \2’ilev, D C. (1981). Snturc (Londor~). 289, 36G373. n’rigley. h’. t:. (1979). Rrit. Xec!. Bull. 35, 35-38. Zcitler. E. 8 Bahr. G. F. (1962). J. Appl. Phys. 33. X47 H.55. Zulauf. Al. Br Eivkr, H. (1979). J. t’hys. C’hun. 83, 380 386. Edited by c’. hzzati