Structural organization of a filamentous influenza A

Lesley J. Calder, Sebastian Wasilewski, John A. Berriman1, and Peter B. Rosenthal2

Division of Physical Biochemistry, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom

Edited* by Robert A. Lamb, Northwestern University, Evanston, IL, and approved April 27, 2010 (received for review February 26, 2010) Influenza is a lipid-enveloped, pleomorphic virus. We combine electron HA trimer in the neutral pH conformation (9), proteolytic frag- cryotomography and analysis of images of frozen-hydrated virions ments of the HA in the low pH conformation (10, 11), and the NA to determine the structural organization of filamentous influenza A tetramer (12, 13) have provided a molecular understanding of the virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous function of these proteins. particles of highly uniform diameter. We show that the matrix layer Further understanding of the virus life cycle requires 3D studies adjacent to the membrane is an ordered helix of the M1 protein and its of ultrastructure that can identify the specific molecular inter- close interaction with the surrounding envelope determines virion actions that govern virus self-assembly. In addition, ultrastructural morphology. The ribonucleoprotein particles (RNPs) that package the changes that are essential to membrane fusion and virion disas- genome segments form a tapered assembly at one end of the virus sembly during cell entry remain to be described. Electron cry- interior. The , which is present in smaller numbers than omicroscopy of influenza virions that are vitrified by rapid plunge- the , clusters in patches and are typically present at the freezing (14–17) shows contrast from virion structures themselves end of the virion opposite to RNP attachment. Incubation of virus at and not from stain distributions, which can also distort or disrupt low pH causes a loss of filamentous morphology, during which we virus structure. However, an understanding of the structural or- observe a structural transition of the matrix layer from its helical, ganization of influenza virus has remained difficult because of its membrane-associated form to a multilayered coil structure inside the pleomorphic nature. Electron cryotomography can be used to virus particle. The polar organization of the virus provides a model for directly determine the structure of pleomorphic virions, although assembly of the virion during budding at the host membrane. Images at a resolution limited by the accumulated radiation damage and and tomograms of A/Aichi/68 X-31 virions show the generality of restricted angles of the tilt series. MICROBIOLOGY fi these conclusions to non- lamentous virions. In the present study, we investigate the structural basis for filamentous morphology in influenza virus. Filamentous mor- electron cryomicroscopy | matrix protein | ribonucleoprotein particles phology is typical of clinical isolates of influenza virus, whereas hemagglutinin | neuramindase a spherical morphology is observed in many laboratory-passaged influenza strains (18, 19). Taking advantage of the surprising nfluenza A virus is a lipid-enveloped orthomyxovirus and a cause structural regularity displayed by an influenza virus (A/Udorn/ Iof human disease by strains that arise through seasonal variation 72) of known, persistent filamentous phenotype (20), we com- and through pandemic infection resulting from viral reassortment bined electron cryotomography of frozen-hydrated virions with or adaptation that introduces new influenza into the human analysis of higher resolution projection images to elucidate the population. The virus genome contains eight separate RNA seg- structural organization of influenza virus. We show that the M1 ments of negative polarity that encode eight structural proteins protein forms a helix associated with the uniform cylindrical and four additional proteins. The genome segments are packaged structure of filamentous Udorn virions. A polar organization of in the virus in complex with the (NP) as ribonu- the virion is evident in both the packaging of the genome and in cleoprotein particles (RNPs) (1). The RNPs are also associated the structure of the surrounding envelope, and applies generally with the viral polymerase (composed of the PA, PB1, and PB2 to influenza virus based on comparison with images and tomo- subunits) (2), which is necessary for viral replication. The virus grams we obtained for the non-filamentous influenza A/Aichi/68 acquires a membrane envelope containing two types of spike gly- X-31. We also studied acid-treated Udorn virus by electron to- coprotein, the hemagglutinin (HA) and the neuraminidase (NA), mography to image virus ultrastructure at the low pH of the by budding through the host plasma membrane. The matrix pro- endosome and observed a structural reorganization of the matrix tein (M1) is the most abundant protein in the virus and forms a layer associated with a change in virus morphology. layer associated with the inside of the envelope. An ion channel (M2) is also present in the virus envelope (3). Results During infection, the virus attaches to cell surface receptors Cryomicroscopy of Frozen-Hydrated Influenza Virions (H3N2) at pH 7. containing sialic acid, which are recognized by the HA protein Fig. 1 shows a gallery of images of both Udorn and X-31 virions (4), and the virus enters the cell by receptor-mediated endocy- aligned by in-plane rotation to emphasize common features. tosis. At the low pH of the endosome, a large structural rear- Udorn virions (Fig. 1 A–G), recorded in a tomogram of a rangement of the HA exposes a hydrophobic fusion peptide and single field of view (tomogram in Movie S1 and Fig. S1A), show mediates fusion of the virus membrane with the host endosomal an extended morphology with capsule-shaped particles (cylin- membrane (4), releasing the packaged contents of the virus (RNPs) into the host cytoplasm. In addition, acidification of the virus core through the M2 ion channel is necessary for mem- Author contributions: L.J.C. and P.B.R. designed research; L.J.C., S.W., and J.A.B. per- brane fusion and release of the RNPs (5, 6). The M1 protein also formed research; S.W. contributed new reagents/analytic tools; L.J.C., S.W., and P.B.R. has an RNP-binding function that regulates transport of RNPs to analyzed data; and L.J.C. and P.B.R. wrote the paper. the where replication and of viral gene The authors declare no conflict of interest. products can occur and subsequently may also transport RNPs to *This Direct Submission article had a prearranged editor. the plasma membrane where virus assembly occurs (7). The NA 1Present address: 51 Oakhill Road, Putney SW15 2QJ, United Kingdom. is a receptor-destroying enzyme that removes sialic 2To whom correspondence should be addressed. E-mail: [email protected]. acid from potential receptors and may have roles in both virus This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. entry and exit (8). High resolution x-ray crystal structures of the 1073/pnas.1002123107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1002123107 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Tomogram sections of frozen-hydrated influenza A virions recorded in a single tomogram field. (A–G) A/Udorn/72 virions from single field shown in Fig. S1A and Movie S1. Virion in A is identical to that in B, but has been manually colored to indicate features corresponding to RNPs (red), multilayered-coil (blue), NA (purple), and HA (green). (K, L, and M) A/Aichi/68 X-31 virions from single field shown in Fig. S1b and Movie S2. Negative stain images of (H)HA rosette (green HA trimer model inset), (I) NA rosette (purple NA head domain tetramer model inset), and (J) RNP purified from X-31. Red lines are used for comparison of RNP length. Purple arcs in D and K indicate typical NA clusters at one end.

drical with a hemispherical cap at each end), typically about 120 by negative stain microscopy (Fig. 1J). These may be tentatively nm in length, and longer filamentous particles, including some assigned to genomic segments 1 and 2, which have the longest over 1 μm in length (Fig. S2). The virions usually have their long sequence. The RNPs only appear to contact the matrix layer at axis in the plane of the thin ice film, but some of the smaller the tapered end of the assembly; they do not interact or make capsule-shaped virions have their axis tilted with respect to the regular contacts with the matrix layer along their length. Some of plane. The filamentous particles show a uniform diameter of the shorter virions are just long enough (120 nm) to package the about 55 nm (measured from the lipid bilayer), whereas the longest, straight RNP segments. average diameter of the shorter capsule-shaped particles is Although the Udorn particles give a clearer view of the extent somewhat wider at 59 nm. There is a tendency for end-to-end and asymmetry of the RNP assembly within the virion, the ar- association of the virions like a string of sausages (Fig. S3). rangement of RNPs within X-31 virions is highly similar (Fig. 1). X-31 virus (Fig. 1 K–M, Figs. S1B and S4, and Movie S2) However, the X-31 virions are shorter than Udorn so that the shows only occasional long filamentous particles, but, compared RNPs extend completely from one end to the other and follow to a previous study by cryomicroscopy (16), we observed that the ellipsoidal profile of the matrix layer and membrane but still a greater fraction are prolate ellipsoids with hemispherical ends. without making regular contacts. Thus, the X-31 RNP assembly Images show 87 out of 145 particles are elongated by a factor appears slightly wider than the Udorn assembly. greater than 1.5 along one axis. X-31 virions are wider (average The observed surface match their known crys- diameter 70 nm, measured from the bilayer) than cylindrical tallographic structures (Fig. 1). The tetrameric NA is slightly Udorn virions. longer (14 nm measured from the membrane) than the HA tri- The virions show an outer membrane containing surface gly- mer (13 nm) and shows less density closer to the membrane coproteins and a dense matrix layer beneath the membrane. consistent with a narrow stalk, for which there is, as yet, no x-ray RNPs are visible inside many virions. The filamentous Udorn crystal structure. Although NAs may be found anywhere on the virions show that the RNPs are part of an assembly held at one virus surface, the tomograms of Udorn virions show that NAs end, and the rest of the interior is typically empty. Some particles cluster at the end of the virion opposite to the end where the appear to lack internal RNP segments yet retain a cylindrical RNPs are attached (Fig. 1). NA clustering at one end is also morphology. Thus, morphology is maintained entirely by the evident in single Udorn images (72 out of 168 virion images have matrix layer and the without requiring RNPs. more than 3 NAs visible at only one end). Tomograms of X-31 The RNPs are part of an assembly that fits just within the inner also show NAs clustered at one hemispherical end of the virion diameter of the matrix layer and tapers as it enters the hemi- (Fig. 1) and single images show clusters (at least 3 NAs) in 44 of spherical cap. In filamentous particles, two RNPs extend further 87 virions, and 25 of these virions show the clusters to be sited at than the others (total length approximately 100 nm) and are one end of the virion. Because molecular overlap can complicate similar in length to the longest purified RNP segments observed the interpretation of structures in projection, clustering at one

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1002123107 Calder et al. Downloaded by guest on September 29, 2021 end is most strongly demonstrated by tomography, and tomo- grams of filamentous Udorn particles now show that the end where clusters are found is opposite to the end where RNPs are attached. Fig. 2A shows an image of an Udorn virion at defocus con- ditions that resolve both the bilayer and the adjacent matrix layer. Lines of density cross the bilayer and are likely to be the transmembrane regions of the HA. Fig. 2 B and C show Udorn virions where bromelain digestion has removed most of the glycoprotein layer. These reveal a clear view of the matrix layer showing thin projections toward the bilayer, which are also evi- dent where it enters the hemispherical caps. Lines of density in the bilayer may be HA transmembrane regions that remain in contact with the matrix layer after glycoprotein removal. Because the distance between glycoprotein ectodomains is such that they do not contact each other, this implies that the spacing of the glycoproteins is attributable to interactions with the underlying matrix layer. Fig. 3. Filamentous influenza A Udorn shows a helical organization of the matrix protein. (A) Image of a virion. (C) Fourier transform of the area within Influenza Udorn Matrix Protein Forms a Helix. The matrix layer is the membrane (black box) in A indicates a helical organization. A lattice a hollow cylinder that appears to have periodic features in images (red) shows the prominent reflections arising from one side of the helix. The and tomograms. Because radiation damage limits the resolution of 38-Å reflection arises from a 7-start helix. (B) Images obtained after two- fi tomograms, we analyzed these periodic features in low dose sided (Upper) and one-sided (Lower) ltering of the transform in C on three layer lines and the equator (indicated by black ticks at right). images (Fig. 3A), which provide higher resolution information in projection. A diffraction pattern (Fourier transformation) (Fig. C 3 ) computed from the image shows that maxima occur along the hemispherical cap regions (Fig. 2 B and C and Fig. S3A). layer lines indicating a helical organization. A strong layer line at These observations are consistent with M1 forming a spherical MICROBIOLOGY 38 Å corresponds to the pitch of a helix slightly inclined from the spiral, but it is not possible to assess the detailed molecular ar- horizontal (although different angles are adopted in different rangement near the poles. Spirals have previously been identified fi images). Images obtained by noise ltering of the transform (Fig. in negative stain studies of disrupted virions (27). 3B) to include data from layer lines only reveal the periodic pattern Fourier transforms of occasional filamentous X-31 virions as associated with the helical symmetry of the cylindrical matrix layer, well as ellipsoidal particles identify a similar 38-Å spacing to the consistent with a subunit of dimensions similar to M1 as derived matrix layer observed in Udorn, although with less order (Fig. S4), from biophysical data and crystal structures for a proteolytic frag- and this can be seen directly in images. The ellipsoidal shape of the ment of the protein (21–26). It has a similar appearance to nega- matrix layer is consistent with a spiral of M1 with a radius de- tive stain images of coils obtained during isolation of the M1 termined by the membrane radius. Virions showing even more protein (26). Thus, the helical component of the matrix layer is an diversity of shape and width still show evidence of a similar, locally organization of the M1 protein, although other viral or host pro- flat 2D lattice of the M1 protein, consistent with M1 being able to teins may associate with the matrix layer. Although a hollow cyl- polymerize with a wide range of curvatures. A previously described inder of about 500-Å diameter, the helix is ordered to at least 12 Å mutant of the M1 protein (K102A) of A/WSN/33, obtained by and suggests a rigid structure. reverse genetics, transforms a non-filamentous strain to a fila- Images recorded at specific defocus values show striations mentous phenotype (28). In Fig. S5, we show that K102A shows from the matrix layer that are limited by the inner radius of the a strong layer line at 38 Å, indicating a similar helical symmetry of membrane and continuously narrow in diameter as they enter the matrix layer to that of Udorn. Thus the helical structure is associated with a specific sequence of the M1 protein.

A Structural Transformation in the Matrix Layer. To study changes in virus structure at the low pH encountered in the endosome, we imaged frozen-hydrated Udorn virions that were acid-treated or acid-treated and then trypsin-treated. Trypsin removes the HA1 residues 28–328 of HA molecules that have undergone the low pH-induced conformational change, which, if present, make it extremely difficult to observe individual glycoproteins. The tri- meric HA2 portion of the molecule that remains after trypsin treatment (10) has previously been shown (29, 30) to be in a con- formation where both its N-terminal fusion peptide and C-termi- nal transmembrane region are inserted into the virus membrane. The ectodomain appears as a thin stalk with a membrane distal knob, quite distinct from both the unchanged HA and the NA. Tomogram sections (Fig. 4 and Movie S3) show a field of acid- treated, trypsin-treated Udorn virus displaying a much wider range of morphologies than is typically seen in untreated pH 7 virus. There are particles with filamentous and spherical shapes, particles of mixed morphology, and some that are likely to be the product of fusion events. The acid-induced change in shape is in Fig. 2. Low-dose images of Udorn virions. (A) Capsule-shaped virion at pH 7 agreement with previous studies, but the filamentous phenotype and (B and C) after bromelain digestion. makes the change appear more dramatic (31). The large spherical

Calder et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 Fig. 4. Tomogram (also shown in Movies S3 and S4) section showing frozen- hydrated influenza A Udorn virions after acid treatment for 10 min at pH 4.9 and trypsin treatment after neutralization.

Fig. 5. Gallery of multilayered coils from acid and trypsin-treated virions. particles likely represent particles that have lost their long fila- (A) Spherical virion with multilayered coil axis perpendicular to image and mentous shape, and the smaller spherical particles are probably resolved membrane. (B) Filamentous particle showing both intact matrix former capsule-shaped particles. In Movie S4, three vesicular layer and matrix layer peeling off membrane to form a multilayered coil. (C) Small coil showing layered structure. (D) Multilayered coil viewed perpen- structures have their membranes distorted into tear-drop shapes dicular to axis showing membrane attachment. (E) Side-on view of multi- by adjacent virions undergoing low-pH associated morphologi- layered coil attached to membrane. (F) Same coil as E viewed down axis. cal changes. When viewing internal virus structures in the tomogram, it becomes evident that the loss of filamentous morphology is as- (Fig. 5 A and C), where the walls of the coil consist of several sociated with disassembly of the highly organized matrix layer layers, each of similar thickness to the helical matrix layer. just beneath the membrane. The image of a spherical virion Fourier transform of projections perpendicular to the coil axis observed in Fig. 5A shows a resolved lipid bilayer containing (Fig. S6) show spacings of 36 Å and 45 Å, similar to the spacings HA2 glycoproteins (low pH-induced conformational change has observed in the 3D packing of the N-terminal domain of M1 in occurred) and no adjacent matrix layer. In contrast, the fila- crystal structures. mentous virion in Fig. 5B has a matrix layer adjacent to and It has been suggested that these dense coiled structures (which almost unresolvable from the lipid bilayer, and most of the HAs are also occasionally apparent in pH 7 virus preparations) are remain in neutral pH conformation. We observe a correlation composed of alignments of the RNP segments (16). In most of the between loss of filamentous shape, disruption of the matrix layer, particles where dense coils are seen in this tomogram, free RNP and conformational change of the HA glycoprotein. segments are clearly visible (Movie S4). The RNPs resemble those The filamentous particles show the matrix layer peeling away seen in filamentous pH 7 particles, and those seen in isolated and from the internal sides of the virions. The spherical particles are purified RNP preparations (Fig. 1J). They are no longer held to- not filled with disorganized matrix layer, rather they possess large gether as a package at one end of the virion. Some do appear to be dense coiled structures, which are usually associated with the associated with the dense coils via one end, but they are separate membrane in small areas where there is still an intact matrix and distinct structures. Disruption of the virus glycoprotein layer layer adjacent to the membrane (Fig. 5 D and E). Where the by bromelain digestion or mercaptoethanol treatment can also matrix layer can be seen detaching from the membrane in the promote the conversion of the matrix layer to multilayered coils filamentous particles, it does so in a fashion that suggests the with identical transform properties (Fig. S7). Images of Udorn association of several strands of the helical matrix layer and particles that are acid-treated without subsequent trypsin treat- a transition into the dense coiled structure observed in spher- ment also show a loss of filamentous shape, disruption of the ical particles. matrix layer, and presence of multilayered, dense coiled structures Similar coil structures have been reported by negative stain (Fig. S8). We cannot discern the shape of individual glycoproteins, microscopy on partially disrupted virions (27) and are not unique presumably due to disorder of the HA1 domains. to the Udorn strain. Single images and tomograms show them to be a coiled structure of varying diameter with a typical pitch of Discussion 110 Å, thick walls and a hollow interior (Fig. 5 D and E). The The structural organization of filamentous influenza virus particles multilayered structure of the coil is most easily viewed when the suggests a model for their assembly. We have shown that a highly axis of the coil is along the vertical direction of the tomogram ordered, helical organization of the matrix layer is associated with

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1002123107 Calder et al. Downloaded by guest on September 29, 2021 the cylindrical morphology of filamentous and capsule-shaped been described before (16, 46), our observation of clustering at Udorn influenza virus. Previously, the M1 gene segment has been the end of the virion opposite the RNPs, close to the point of identified as a genetic determinant of filamentous morphology (20, release from the host and in an orientation such 28, 32, 33). Although the M1 protein alone was reported to be that the neuramindase active site is positioned to hydrolyze essential for virus-like particle (VLP) formation and capable of receptors on the host cell surface, may reflect a localized re- forming intracellular tubes (34), it now appears that viral mem- quirement for receptor destruction (46). Inhibitors of NA en- brane proteins are required for budding (35). A helix provides zyme activity result in a virus that is stalled or aggregated at the a specific structural mechanism by which M1 subunits can be added host membrane. NA inhibitors that are anchored to the mem- to a rigid assembly that drives budding at the plasma membrane. brane could target this site of action. Regular interactions between the matrix layer and membrane In viruses incubated at the low pH of the endosome, there is components suggest that recruitment of membrane glycoproteins a correlation between loss of filamentous shape, disassembly of couples M1 helix formation with budding through the membrane. the matrix layer, and conformational change of the HAs. We Images of filamentous influenza particles budding from cells, observe that the helical matrix layer dissociates from the virus obtained by electron microscopy of stained, plastic sections, membrane at low pH, forming an alternative, stable assembly suggest that an RNP assembly is found on the apical end of described here as a multilayered coil. We also find intermediate a budding virion and distal to the virus membrane (36). It is likely structures in this coiling transition. In addition to the M2 ion that the RNP assembly may be an important trigger for budding. channel, acidification of the virus interior could be a conse- The M1 helix is nucleated near the point of attachment of the quence of insertion of the fusion peptide of HA into the virus RNP assembly and the subsequent polymerization process drives membrane. Because the multilayered coil can also form at filamentous virion budding. RNPs may influence the width of the neutral pH, particularly when the glycoproteins are disrupted, surrounding matrix layer and also determine the minimum low pH is not a requirement for their formation, but rather they length of the virion before pinching-off can occur. are a consequence of concerted dissociation of the matrix layer The sequence of the Udorn M1 protein likely confers the fil- from the membrane. Disassembly of the matrix layer at low pH amentous phenotype by stabilizing specific helical contacts in the may facilitate membrane fusion by exposing bare patches of matrix layer (28). Length variation in Udorn particles may reflect membrane bilayer or by allowing freedom of movement of the a competition between extension of the M1 helix and the com- glycoproteins to form fusogenic complexes. Acidification of the pletion of budding (“pinching off”) and release. The “beaded” virion interior through the M2 ion channel increases the rate of

structures that result from the end-to-end association, reminis- membrane fusion between influenza viruses and liposomes (5). MICROBIOLOGY cent of budding defects in other lipid-enveloped viruses (37), Matrix disassembly and the associated morphological changes may reflect this competition. Cellular factors associated with the also facilitate release of individual RNP segments from the as- vacuolar sorting pathway are implicated in virus budding and sembly attached at the apical tip of the virion. release for several virus families, and some of these may form protein assemblies that regulate the physical constriction re- Materials and Methods quired for pinching-off (38). It is not known what host factors Growth and Purification of Virus. A/Udorn/72 (a gift from R. Compans, Emory regulate orthomyxovirus budding (39, 40), but host factors such University School of Medicine, Atlanta, GA), A/WSN/33, and M1 mutant as actin and the virus M2 protein may contribute to the fila- K102A (a gift from D. Steinhauer, Emory University School of Medicine, mentous phenotype (20, 41, 42). Atlanta, GA) were grown in Madin-Darby canine kidney (MDCK) cells and The ellipsoidal shape of the typical X-31 matrix layer may be purified over sucrose gradients as previously described (28). A/Aichi/68 X-31 thought of as two spiral structures similar to those in the hemi- virus was grown in embryonated hens’ eggs and purified over sucrose gra- spherical caps of the Udorn virions but without the intervening dients as previously described (47). cylindrical region. The shape of the X-31 matrix layer reflects the Low pH Incubation and Trypsin Treatment of Virus. Virus was taken to pH 4.9 absence of strong intrinsic helical or cylindrical polymerization of with 0.1 M sodium citrate and incubated at 20 °C for 10 min and then the X-31 M1 protein due to sequence differences to Udorn M1 fl neutralized with 1 M Tris. In the case of further trypsin treatment, virus was and may be in uenced by the shape of the RNP assembly or the then incubated with trypsin (Sigma) at an enzyme:protein ratio of 1:50 for 1 membrane. The M1 protein’s capacity to polymerize in 2D lat- h at 20 °C, before stopping the reaction with trypsin inhibitor (Sigma). Virus tices with a wide range of curvature is an important basis for was then pelleted and resuspended in PBS. influenza virus pleomorphy and reflects the use of a compara- tively small building block to build a structure with a large radius. Bromelain Digestion of Virus. Virus in 0.1 M Tris buffer pH 8.0 was incubated The budding driven by polymerization of the M1 helix may with bromelain (Sigma) at an enzyme: ratio of 1:2 together with lead to the observed segregation of HA and NA in the viral 50 mM β-mercaptoethanol for 3 h at 37 °C. A further aliquot of bromelain membrane, either by recruiting HA through specific contacts and mercaptoethanol was added (at the same enzyme:viral protein ratio) between M1 and the HA cytoplasmic domain or by exclusion of and the sample incubated at room temperature for 18 h; 40 mM iodoace- tamide was added to stop the digestion and the virus pelleted (TL100 cen- NA clusters that display lateral interactions between head- I trifuge, 55,000 rpm, 10 min) and resuspened in PBS. Control virus samples domains (Fig.1 , NA rosettes). NA clusters may lead to pinching- were also treated under identical conditions with either iodoacetamide or off by uncoupling matrix layer extension from glycoprotein re- mercaptoethanol in the absence of bromelain. cruitment and bud extension. The cytoplasmic domains of the HA and NA can both influence particle shape (43, 44). Mem- Electron Cryomicroscopy. Four microliters of virus sample was mixed with 10- brane microdomains such as “rafts” are thought to concentrate nm gold particles (British-Biocell) and applied to amylamine glow-discharged the HA at the sites of budding (45). VLPs have also been 200 mesh copper Quantifoil (R2/4) grids in the environment chamber (4 °C, reported with expression of the HA and NA in the absence of the 90% RH) of a Vitrobot Mark III (FEI), blotted on both sides with a double layer matrix protein (35), but we assume these cannot possess the axial of paper for 4 s before plunging into liquid ethane. Grids were transferred to a Gatan 626 tomography holder or Polara stage. Imaging was performed in organization driven by M1 helix polymerization and glycoprotein fi recruitment as described here. an FEI Spirit TWIN microscope at 120 keV using a tungsten lament source and equipped with a cryobox around the sample. Images were recorded The organization of glycoproteins in the virus envelope may be fi unbinned on an Eagle 2K camera (FEI) at 21 K (10.0 Å/pixel), 30 K (7.2 Å/pixel), of functional signi cance. The rigidity, radius of curvature, and or 52 K (4.3 Å/pixel) magnification under a range of defocus conditions. regular spacing of the HA glycoprotein lattice may provide Magnification was calibrated by recording images of tobacco mosaic virus specific geometric constraints on receptor-binding and mem- under identical conditions. Tilt series for tomography were recorded as in- brane fusion. Although clustering of NAs on the virus surface has dividual low-dose images or automatically using Inspect3D (FEI) from 0 to ±

Calder et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 construction procedure using the Priism package (49). Images were analyzed 66° in 3° or 4° steps, typically with a total dose less than 50 e-/Å2. An FEI G2 fi Polara operating at liquid nitrogen temperature and 300 keV was used to using FFTRANS and Ximdisp programs from the MRC package (50). The l- collect images and tilt series on a 224 HD detector (TVIPS) at 15 K (9.1 Å/pixel), tered images (Fig. 3B) were obtained by masking transforms (program or on film at 59 K magnification, which was digitized at 7 μm/pixel on a Z/I TRMSK) to include peaks from both sides of the indicated layer lines or from scanner densitometer. one side corresponding to points on the lattice (Fig. 3C) and the equator. In the tomogram in Movie S2, gold particle fiducials were computationally Image and Tilt Series Analysis. Tomographic tilt series were aligned using removed from aligned tilt series before reconstruction. IMOD software (48). Alignment initially used cross-correlation and then used gold particles or other dense features as fiducials. Reconstructed 3D volumes ACKNOWLEDGMENTS. This work was funded by the Medical Research Coun- were generated by back-projection or by an iterative alignment and re- cil (United Kingdom) under program code U117581334.

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