Influenza virus pleiomorphy characterizedby cryoelectron tomographyAudray Harris*, Giovanni Cardone*, Dennis C. Winkler*, J. Bernard Heymann*, Matthew Brecher†,Judith M. White†, and Alasdair C. Steven*‡

*Laboratory of Structural Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health,Bethesda, MD 20892; and †Department of Microbiology, University of Virginia, Charlottesville, VA 22908

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved October 13, 2006 (received for review September 7, 2006)

Influenza virus remains a global health threat, with millions ofinfections annually and the impending threat that a strain of avianinfluenza may develop into a human pandemic. Despite its impor-tance as a pathogen, little is known about the virus structure, inpart because of its intrinsic structural variability (pleiomorphy): theprimary distinction is between spherical and elongated particles,but both vary in size. Pleiomorphy has thwarted structural analysisby image reconstruction of electron micrographs based on aver-aging many identical particles. In this study, we used cryoelectrontomography to visualize the 3D structures of 110 individual virionsof the X-31 (H3N2) strain of influenza A. The tomograms distin-guish two kinds of glycoprotein spikes [hemagglutinin (HA) andneuraminidase (NA)] in the viral envelope, resolve the matrixprotein layer lining the envelope, and depict internal configura-tions of ribonucleoprotein (RNP) complexes. They also reveal thestems that link the glycoprotein ectodomains to the membrane andinteractions among the glycoproteins, the matrix, and the RNPsthat presumably control the budding of nascent virions from hostcells. Five classes of virions, four spherical and one elongated, aredistinguished by features of their matrix layer and RNP organiza-tion. Some virions have substantial gaps in their matrix layer(‘‘molecular fontanels’’), and others appear to lack a matrix layerentirely, suggesting the existence of an alternative budding path-way in which matrix protein is minimally involved.

envelope glycoproteins � matrix protein � ribonucleoprotein particles �virus assembly � virus structure

Influenza virus belongs to the orthomyxoviridae, a family ofenveloped viruses with segmented genomes of single-stranded

negative-sense RNA (1). Although high-resolution structures havebeen determined by x-ray crystallography for several viral compo-nents or fragments thereof (2–4), information about the 3D struc-ture(s) of complete virions has remained scanty. Current models,based primarily on electron microscopy of negatively stained sam-ples (5–7), envisage an envelope containing the trimeric hemag-glutinin (HA), tetrameric neuraminidase (NA) (8), and M2 (9)glycoproteins, lined with a continuous layer of matrix protein,enclosing multiple ribonucleoprotein (RNP) complexes (10). AnRNP complex consists of a segment of genomic RNA coated by thenucleocapsid protein, with a loop at one end, whereas the other endhas a duplex formed by base pairing of the termini to which the viralpolymerase is bound (11–14).

Influenza virions assemble as they bud from the surface ofinfected cells. In this process, the glycoproteins accumulate in lipidrafts, where they interact with the underlying matrix protein. Hostfactors also participate, particularly in the final stage of pinching off(15). During assembly, a dilemma common to all viruses withsegmented genomes must be resolved: how to assign the varioussegments appropriately to nascent virions? In transverse thin-section electron micrographs of budding influenza virions, a com-monly observed motif is a ring with seven features, each thought tobe an RNP, surrounding a central such feature (16, 17). Their totalnumber, eight, matches the number of distinct RNA segments in theinfluenza virus genome. However, it has not been determined

whether each virion receives one copy of each segment or a randomselection, nor how RNPs are recruited to the budding site (15).

To date, influenza virus has been refractory to 3D structuralanalysis, in large part because its pleiomorphy has precludedvisualization by image reconstruction of electron micrographs byprocedures that rely on averaging many identical particles, whichhave been applied successfully to many icosahedrally symmetricviruses (18). The virion structure is of interest not only in thecontext of virus assembly but also in light of the possibility thatpleiomorphic variations may correlate with infectivity and/or patho-genicity. As a step toward addressing these questions, we have usedcryoelectron tomography, a technique capable of rendering the 3Dstructures of individual macromolecular particles in their nativestates (19–25), to visualize influenza virions of the type A egg-adapted X-31 strain (26).

ResultsInfluenza Virus Morphology. Density maps of 110 virions werecompiled from six tomograms. Their resolution is 5.5 nm in-plane,according to the NLOO2D criterion. A central slice through one ofthe tomograms is shown in Fig. 1. The majority of virions (n � 78)may be described as spherical (axial ratio �1.2), the remainder areoval or kidney-shaped with axial ratios up to 1.4 (n � 17) or moreelongated with axial ratios as high as 7.7 (n � 15). The outerdiameters of the spherical virions ranged from 84 to 170 nm (mean,120 nm). The average short diameter of elongated virions was fairlyuniform and lower, at �100 nm.

Despite their pleiomorphy, most virions have three features incommon. First, they are covered with glycoprotein spikes, protrud-ing from the virion surface. Second, underlying the spikes at thevirion periphery are two layers of approximately equal density andthickness. These layers are closely apposed with a center-to-centerspacing of 5.5 nm. We interpret the outer layer as the lipid bilayerin which the transmembrane segments of the glycoproteins areimplanted and the inner layer as a monolayer of matrix protein.Third, they contain RNPs whose appearance varies according totheir disposition relative to a given tomographic slice, but whichtypically present as curved or straight filaments (arrowheads, rightside, Fig. 1) or, in cross-section, as C-shaped densities (arrowheads,left side, Fig. 1).

Based on their appearance in tomograms, virions were sortedinto five classes, I–V, listed according to relative abundance: I (n �88), II (n � 15), III (n � 5), IV (n � 1), and V (n � 1) (Fig. 2). ClassII comprises elongated particles; the other four classes emergedfrom further sorting of the spherical particles. The criteria applied

Author contributions: A.H. and G.C. contributed equally to this work; A.H., J.M.W., andA.C.S. designed research; A.H., G.C., D.C.W., J.B.H., and M.B. performed research; G.C. andJ.B.H. contributed new reagents/analytic tools; A.H., G.C., D.C.W., and J.B.H. analyzed data;and A.H., G.C., and A.C.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: NA, neuraminidase; RNP, ribonucleoprotein.

‡To whom correspondence should be addressed. E-mail: stevena@mail.nih.gov.

© 2006 by The National Academy of Sciences of the USA

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were the presence or absence of a discernible matrix layer and RNPorganization. Class I has an evident matrix layer and a somewhatdisordered arrangement of RNPs; class III lacks a discerniblematrix layer and also has jumbled RNPs. Classes IV (with matrix)and V (no matrix) have condensed cores that appear in longitudinal

slices (Fig. 2 d and e) as parallel arrays of seven to eight rods;however, tracking them through the tomograms revealed that thesecores are single solenoids of seven to eight coils with a pitch of �10nm (Fig. 7c). The contour length of the coil was measured on a classV virion to be �1.26 �m. The elongated class II particles share thefeatures of class I, except that their RNPs tend to be approximatelyaligned with the long axis of the particle (Fig. 2b). The possibilityremains that elongated counterparts of the rarer classes III, IV, andV may be detected in a larger sampling of virion populations.Indeed, in cryomicrographs, we did observe some elongated virionswith solenoid-like contents (data not shown).

Glycoprotein Spikes. Except in a few low-density patches (e.g., arcs,Fig. 1), the glycoproteins are packed closely but irregularly, with anaverage center-to-center spacing of �11 nm. Both HA and NAextend radially from the membrane to terminate in bulbous heads(Fig. 3a). NA is slightly longer than HA and may be distinguishedin longitudinal sections by its shorter head (HA has a characteris-tically bilobed ‘‘peanut’’ shape) and longer stem (Fig. 3 b vs. c) andin transverse sections by its square profile, as opposed to thetriangular profile of HA (Fig. 4).

The packing density of spikes was assessed by counting themwithin typical well resolved patches; on average, an area of 40 � 40nm was found to contain 13 spikes. No difference in this respect wasobserved between spherical and elongated virions. Thus a sphericalvirion of average diameter (120 nm) should have �375 spikes. Thisfigure was confirmed by counting all of the spikes on two virions ofaverage diameter whose entire surfaces were visualized clearlyenough for this to be possible, yielding 351 (301 HA plus 50 NA)and 328 (290 HA plus 38 NA) spikes per virion. Allowing for a fewbare patches (such patches were observed on �40% of sphericalvirions, e.g., Fig. 4 b–d), these quantitations are consistent. They arealso in reasonable agreement with prior biochemical estimates of340–400 HA trimers per virion (10).

We used molecular modeling to exploit our ability to identifyindividual spikes in the tomograms as HA or NA (Fig. 3). The HAectodomain solved by x-ray crystallography is �12 nm in depth (2)

Fig. 1. Section through a cryotomogram of a field of influenza virions. Three planes were averaged to reduce noise, giving a section 4.7 nm thick. Whitearrowheads mark typical RNPs: at left, transverse C-shaped sections; at right, longitudinal sections through relatively straight as well as curved RNPs. Black arcsmark areas with matrix layer gaps and missing or lower density packings of glycoprotein spikes. The framed virion is shown in greater detail in Fig. 2a. The largeirregular particle at top right was probably generated during cell disruption; it contains at least one budding virus (white asterisk). (Scale bar, 100 nm.)

Fig. 2. Examples of the five morphological classes (I–V) of influenza virions.For the more abundant class I and II particles, three serial slices, 10.9 nm apartand 4.7 nm thick, are shown in a and b, respectively, whereas single near-central slices are shown for classes III-V in c–e. The class I virion (a) is the framedparticle in Fig. 1. (Scale bar, 50 nm.)

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and that of NA, �6 nm (ref. 3; Fig. 3 b and c). When the HAectodomain is docked into tomographic densities for HA spikes, itsouter tip is �14 nm from the membrane surface (i.e., there is a 2-nmstem), whereas for NA spikes, this spacing is �16 nm (i.e., a 10-nmstem). Between the ectodomain sequences and the predictedtransmembrane regions are linkers of 10 (HA; ref. 27) and 51 (NA;ref. 28) amino acids, respectively. If they are predominantly �-he-lical, this would correspond to stem lengths of 1.5 and 7.6 nm,respectively, in general accord with our measurements.

The distributions of HA and NA over the virion surface appearnot to be entirely random; congregations of the more abundant HAspikes are the pattern most frequently observed; then single NAspikes surrounded by HA, and local clusters of NA (Fig. 3a). A

tomographic model showing the distribution of spikes over thesurface of a single virion is presented in Fig. 3d.

Matrix Protein Layer. In virions of classes I, II, and IV, a matrix layeris visible under the viral membrane and closely apposed to it (Figs.1–3). Their center-to-center separation is �5.5 nm, close to ourresolution limit. The matrix layer has the same apparent thicknessas the membrane. Contacts between them, presumably by theconnecting densities that are seen in the tomograms (e.g., Fig. 3 aand b) but are at our resolution limit, should involve interactionsof the matrix protein with the small endodomain tails of theglycoproteins (29), predicted sizes, 6 (NA; ref. 28) and 10 residues(HA; ref. 27). The existence of direct interactions of the glyco-proteins with the matrix layer is further supported by the correlationof gaps in the matrix layer with membrane regions in which theglycoproteins are either absent or less abundant than elsewhere(Fig. 5 a and b).

Of the particles with a matrix layer (102/110), �20% containeda visible gap. This is probably an underestimate, because gapsoriented perpendicular to the untilted specimen plane should beless reliably detected because of lower resolution in this dimensioncaused by the incomplete tilt range covered (the so-called ‘‘missingwedge’’ effect; ref. 30). Some particles had more than one gap;specifically, 13 had 1 gap, 5 had 2 gaps, and 2 had 3 gaps. The gaps

Fig. 5. Influenza virus particles with gaps in their matrix layer. Virionscontaining matrix gaps tend to have a decreased density of glycoproteins inthe envelope regions overlying the gaps, which are indexed with arcs. Thewhite arrowheads in d points to C-shaped transverse sections through twoRNPs. For each particle, a 4.7-nm-thick slice is shown. (Scale bar, 50 nm.)

Fig. 3. Distributions and shape-based differentiation of HA and NA spikes.(a) HA cluster (Left); single NA (marked) in a cluster of HAs (Center); and clusterof mainly NA spikes (Right). (Scale bar, 50 nm.) (b and c) The stem lengths ofHA and NA (square brackets in b and c, respectively) were measured asdescribed in Materials and Methods. The structures of the stems, transmem-brane segments, and endodomain tails are not known, and they are shownschematically. Molecules in the matrix layer are inferred to be packed in amonolayer with a spacing of �4 nm (our unpublished results). (Scale bar, 5nm.) (d) Model of the distribution of glycoprotein HA (green) and NA (gold)on a single influenza virion. The lipid bilayer is blue. (Scale bar, 20 nm.)

Fig. 4. Patches of glycoprotein spikes depicted in tangential sections (4.7 nmthick) through virions in cryotomograms. In most cases, triangular HA spikes(e.g., white arrowhead in a) are distinguishable from square NA spikes (e.g.,white arrowhead with black border in a). Spikes are close-packed, withoutlateral order, apart from occasional bare patches of variable size. Someexamples of bare patches are shown in b–d. (Scale bar, 20 nm.)

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ranged in size up to �80 nm. Virions of classes III and V, amountingto 6% of the data set, appear to lack a matrix layer entirely (Fig. 2c and e).

RNPs. The RNPs are packed quite densely in most virions, makingit difficult to delineate individual complexes. Nevertheless, we couldmake the following observations, based on the more abundant classI and II particles (Fig. 2 a and b). RNPs are �14 nm thick, and insome cases, mostly in class II virions (Fig. 2b), we were able to traceindividual complexes, whose lengths were found to vary from �24to �110 nm (n � 40). Each RNP makes contact with the matrixlayer at one or both ends. Most RNPs exhibit some curvature. Theytypically present a C-shaped motif in cross-section (e.g., arrow-heads, left side; Figs. 1 and 5d). This motif tends to rotate betweensuccessive sections, suggesting a loosely ordered but basically helicalstructure (ref. 11; Fig. 6).

The arrangement of the RNPs varies with the morphology andsize of the virion. In elongated particles, they form a near-parallelbundle in which all RNPs associate with the matrix at the same endof the virion (Fig. 2b). In a few instances, we were able to distinguisheight such complexes, usually one at the center surrounded byseven. Some spherical particles exhibit a similar arrangement in atleast one plane (e.g., Fig. 7 a and b), albeit with the RNPs not sowell aligned. Smaller particles (�100 nm in diameter; �10% of thedata) tend to have fewer than eight segments. The minimumnumber detected was two. In large spherical particles (�130 nm),the arrangement of RNPs was less orderly. In spherical particles, thecontact points of the various RNP complexes on the matrix layerwere not clustered but were distributed around the interior surface.

DiscussionThe present observations afford insight into the molecular archi-tecture of influenza virions, whose diverse forms represent theoutcome of complex patterns of interactions among viral compo-nents: the glycoproteins, matrix protein, and the RNPs; and hostfactors (15). Because different viral strains propagated in the samecells produce different distributions of virion shapes (e.g., elongatedvs. spherical), it follows that plasticity in the interactions among viralcomponents is a major contributor to pleiomorphy. In this study, wehave used electron tomography to make a systematic account ofpleiomorphic variability in one well studied strain of the virus.

The Matrix Layer. In assembly, the matrix protein has the dual rolesof concentrating the glycoproteins at the budding site (there appear

to be no regular interactions between neighboring spikes; Fig. 4)and of interacting with the virion’s complement of RNPs. If, asseems likely, the matrix layer involves a regular packing of subunits(ref. 7; our unpublished observations), this packing must be adapt-able enough to allow the formation of differently sized and shapedvirions.

Some virions have substantial gaps in their matrix layer. Wesuspect that these ‘‘molecular fontanels’’ may represent sites wherethe virions pinched off in budding from the host cell. It is notapparent whether the gaps have functional significance, but theclose association of the matrix protein with the membrane suggeststhis association might impede membrane fusion, and a matrix-freearea of envelope might offer a favorable site to initiate the fusionprocess.

We also observed some virions, classes III and V, that appear tolack a matrix layer entirely (Fig. 2 c and e). This observation impliesthat either they have only a few molecules of matrix protein, enoughto play its role in budding but not enough to be perceived as acontinuous layer in the tomograms, or they assemble by a differentbudding mechanism.

We have considered some possible relationships between spher-ical virions and elongated ones; for instance, that spherical virionsmay bud in the same way but simply have pinched off earlier.However, this hypothesis does not square with the observation thatmost spherical virions have larger diameters (120 vs. 100 nm).Alternatively, could spherical virions be derived from elongatedones by an in vitro rearrangement in overall structure accompaniedby a disordering of the RNPs? This hypothesis, although not ruledout, invokes a major reorganization of the matrix layers, afterbudding.

Fig. 7. Central slices through influenza virions containing (7 � 1)-like andsolenoid RNP configurations. a and b show two examples of (7 � 1)-like config-urations in spherical class I virions. In each case, a slice is shown in Left and, onRight, it is indexed with red rings around the RNPs, which are mostly C-shaped incross-section. In virion (a), which has an incomplete matrix layer, only six periph-eral RNPs are clearly visible (two are enclosed in the red ellipse). Weaker densitymay denote a seventh peripheral RNP. (c) Two serial slices through the solenoidalcore of a class V virion, which has no discrenible matrix layer. The slices are 18.7nm apart and 4.7 nm thick. (Scale bars, 50 nm.)

Fig. 6. Distributions of density perpendicular to and along influenza virusribonucleoprotein particles are illustrated in tomographic slices. (a–d) Four serialslices, 4.7 nm thick and 9.4 nm apart, through part of an influenza virion. An RNPsegment is oriented approximately perpendicular to the viewing direction. (e) Acentral longitudinal section of the same RNP complex, after computationalstraightening, suggests a double helical structure. (Scale bar, 20 nm.)

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RNPs. EM studies of stained plastic sections of budding influenzavirions have presented evidence for a (7 � 1) configuration ofaligned RNPs (17). Our observations of complete ice-embeddedelongated virions (class II) are consistent with this model. Althoughwe observed that smaller virions tend to have fewer than eightRNPs, we also observed some spherical virions whose RNPs weresimilarly arranged (e.g., Fig. 7 a and b). More commonly, we couldcount eight RNPs, but they were not aligned. These observationssuggest there may be specialized attachment points for each RNPon the matrix layer, with these points being closely clustered or evenmerged in elongated virions and less regularly arranged in sphericalvirions. However, we have yet to detect structural features in thematrix layer that are specific to these sites.

The condensed core of our class IV and V particles (Fig. 2 d ande) has been observed before, also in rare occurrence (31). Ourtomograms reveal that these cores are solenoidal windings of afilament that is much longer than any individual RNP that weobserved and whose diameter is about half that of an RNP. Onepossible explanation is that the solenoid may represent an immatureprecursor form in which the RNPs are initially packaged as a singleconcatenated filament that is subsequently dispersed and reorga-nized. Although speculative, this proposition would afford a mech-anism whereby a designated consignment of multiple segmentscould be incorporated into a given viral particle.

Materials and MethodsVirus Propagation and Purification. X-31 [A/Aichi/68 (H3N2)] in-fluenza virus, grown in embryonated chicken eggs and step gradi-ent-purified according to standard procedures, at �2 mg/ml pro-tein, was purchased from Charles River Laboratories (NorthFranklin, CT). For electron microscopy, the virus was furtherconcentrated by pelleting (137,000 � g for 2 h) and resuspended at�6 mg/ml protein in 20 mM Hepes/20 mM Mes/130 mM NaCl, pH7.5.

Tomographic Data Collection. Virus was mixed 1:4 with a suspensionof 10-nm colloidal gold particles (Ted Pella, Redding, CA). Four-microliter drops were applied to holey carbon films, thinned byblotting, and vitrified by plunging into liquid ethane before transferinto a liquid-N2-cooled specimen holder (model 626; Gatan, War-rendale, PA). A Tecnai-12 electron microscope (FEI, Hillsboro,OR) operating at 120 keV was used to record single-axis tilt seriesat 1° steps, covering ranges of approximately �64° to �72°. Themicroscope was equipped with an energy filter (Gatan GIF 2002)that was operated in the zero-energy-loss mode with a slit width of

20 eV. Images were recorded on a 2,048 � 2,048-pixel CCD camera(Gatan) at �38,500 magnification (0.78-nm pixels) and 4–6 �mdefocus, corresponding to first zeros of the contrast transfer func-tion at (3.7 nm)�1 � (4.5 nm)�1. Data were recorded underlow-dose conditions, using SerialEM to conduct automatic tilting,tracking, focusing, and image acquisition (32). The cumulativeelectron dose was �55 electrons/Å2.

Image Processing. Projections in a tilt series were mutually alignedby using the gold particles as fiducial markers and the tomogramcalculated by the weighted back-projection method, using IMOD(33). The final analyses were performed on binned tomograms witha voxel size of 1.56 nm. The tomograms were denoised by bilateralfiltering (34), with the following parameters: 1 iteration; �1 � 1pixel; �2 � 3–5 times the standard deviation of the tomogram; anda kernel size of 5. These parameters were chosen to provide amoderate reduction in noise, while preserving information atrelatively high resolution. Their resolution was estimated by theNLOO-2D method as implemented in ELECTRA (35). For eachtomogram, resolution was assessed as the average of three particlesat the threshold 0.3. Cryoelectron microscopy of virions was per-formed as reported (36).

Mapping the Distribution of Glycoprotein Spikes. The lengths of theglycoprotein stems were measured after aligning their respectiveectodomain crystal structures and a model lipid bilayer, taken to be4 nm thick, with the corresponding densities in the tomograms. Tofacilitate processing of the glycoprotein layer, the viral membraneand the extraviral region were masked out. A model was generatedin which each pair of pseudoatoms was connected by a pseudobondfitted by hand into each density identified as a glycoprotein.Templates of the ectodomains were generated at 5.0-nm resolutionfrom the crystal structures (3HMG for HA and 1V0Z for NA). A3-nm-wide stalk was added to the NA map. These templates wereused to refine the positions of the glycoproteins by crosscorrelation(taking into account the missing wedge), as well as to classify themas HA or NA, and were also used to build a model of theglycoprotein layer. This processing was done in Bsoft (37). Visu-alization was done with Chimera (www.cgl.ucsf.edu/chimera).

We thank Charles S. Smith and Brittney Manvilla for help with electronmicroscopy. This work was supported in part by the Intramural ResearchProgram of National Institute of Arthritis and Musculoskeletal and SkinDiseases and the National Institutes of Health (NIH) Intramural Tar-geted Antiviral Program (A.C.S.) and an NIH R01 award, AI22470 (toJ.M.W.). M.B. was supported in part by an NIH training grant(5T32AI07047).

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