Electron Cryotomography of Measles Virus Reveals How Matrix Protein Coats the Ribonucleocapsid Within Intact Virions

Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions

Lassi Liljeroosa, Juha T. Huiskonenb, Ari Oraa, Petri Susic, and Sarah J. Butchera,1

aInstitute of Biotechnology, University of Helsinki, 00790, Helsinki, Finland; bOxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom; and cDepartment of Virology, University of Turku, 20520, Turku, Finland

Edited by Michael G. Rossmann, Purdue University, West Lafayette, IN, and approved September 20, 2011 (received for review April 18, 2011)

Measles virus is a highly infectious, enveloped, pleomorphic virus. been limited mainly by the substantial pleomorphism and large We combined electron cryotomography with subvolume averag- size of the viral particles. To date, Sendai virus is the only ing and immunosorbent electron microscopy to characterize the member of the Paramyxoviridae for which a 3D structure of the 3D ultrastructure of the virion. We show that the matrix protein virion has been described (14). For MV, such studies have not forms helices coating the helical ribonucleocapsid rather than been performed, but two differing ultrastructural models can be coating the inner leaflet of the membrane, as previously thought. proposed (Fig. 1). In the first, commonly used model (15), The ribonucleocapsid is folded into tight bundles through matrix– M remains bound to the membrane after budding, leaving the matrix interactions. The implications for virus assembly are that nucleocapsid largely free inside the particle (Fig. 1A), similar to the matrix already tightly interacts with the ribonucleocapsid in the Sendai virus structure (14). In the second model, M mainly covers the nucleocapsid, creating a regularly packed form effi- the cytoplasm, providing a structural basis for the previously ob- fl served regulation of RNA transcription by the matrix protein. Next, cient for genome packaging and budding that could in uence the matrix-covered ribonucleocapsids are transported to the viral RNA synthesis (Fig. 1B). This model is based on observa- plasma membrane, where the matrix interacts with the envelope tions that M is a common contaminant of nucleocapsid prepa- glycoproteins during budding. These results are relevant to the rations (16) and early electron microscopy studies, which have nucleocapsid organization and budding of other paramyxoviruses, described two distinct types of tubular structures with different diameters inside MV-infected cells (17–21), where the larger of where isolated matrix has been observed to form helices. the two tubes has been reported to contain M (20). Although the structure of recombinant helical nucleocapsids (∼20 nm in dia- image reconstruction | subtomogram averaging meter) has been well characterized (22–24), the in situ organization of the different structural components in measles virions easles is a common, acute disease caused by measles virus is unresolved, as illustrated by the two hypotheses presented in M(MV), which is one of the most infectious viruses known. Fig. 1. The resolution of the structural organization will have The symptoms include fever, conjunctivitis, respiratory infection, a direct impact on our understanding of MV assembly. and maculopapular rash, and the disease is severe, particularly Here we have used cryo-EM to characterize the 3D ultra- among children and in immune-compromised patients (1). The structure of purified MV particles. We observed both bare and high mortality of measles is often associated with secondary covered nucleocapsids in the virion, supporting the model pre- bacterial infections that take place during the period of virus- sented in Fig. 1B. Tomographic subvolume alignment and av- induced immunosuppression. There has been an effective vac- eraging allowed us to describe the 3D organization of these two cine against the virus since the 1960s, but because of poor concentric helical structures inside the virions. In addition, by vaccination coverage in many developing countries the virus still using immunosorbent electron microscopy (EM), we show that remains a major problem. In 2008, measles caused 164,000 the inner helix contains N and the outer helix contains M. The MICROBIOLOGY deaths worldwide, being one of the leading causes of death matrix-covered nucleocapsids were seen to form tight bundles. among young children (2). Taken together, these data reveal MV organization and further MV belongs to the genus Morbillivirus in the family of Para- deepen our understanding of MV maturation. myxoviridae that also includes other major human pathogens, such as mumps virus, respiratory syncytial virus (RSV), and the Results parainfluenza viruses. All of the paramyxoviruses are enveloped viruses that enclose a helical nucleocapsid composed of the Measles Virions Are Highly Pleomorphic, Covered by the Surface negative-stranded ssRNA genome and nucleoprotein (N), along Glycoproteins to a Varying Extent, and Contain Two Types of Tubular with the matrix protein (M), the phosphoprotein (P), and the Structures Inside. Because conventional EM methods do not large polymerase protein (L). In addition, MV has two mem- standardly preserve high-resolution information for the 3D re- brane-spanning glycoproteins, fusion (F), and an attachment construction of pleomorphic enveloped particles, we used cryo- protein, hemagglutinin (H), on the surface. These two proteins EM to look at measles virions in their native hydrated state are responsible for the binding and entry of the virus into host (25, 26). We had two strains available, a WT and an Edmonston cells (3). The M protein of paramyxoviruses is thought to co- ordinate assembly of the virion and to form a thin layer bound to the inner leaflet of the virion membrane. For MV, interactions of Author contributions: L.L., A.O., P.S., and S.J.B. designed research; L.L., J.T.H., and A.O. the M protein with the cellular membrane (4–6), with the performed research; L.L., J.T.H., and S.J.B. analyzed data; and L.L., J.T.H., A.O., P.S., and N protein (6–9), and with the tails of the two surface glyco- S.J.B. wrote the paper. proteins (10–13) have been described. The interaction with the The authors declare no conflict of interest. N protein has been shown to be mediated by two leucine residues This article is a PNAS Direct Submission. at the C terminus of the intrinsically unstructured C-terminal Data deposition: The average models reported in this paper have been deposited with the domain of N (NTAIL). This interaction was shown to be crucial Unified Data Resource for 3D Electron Microscopy, EMDatabank.org (accession codes for both recruiting the nucleocapsid to the plasma membrane EMD-1973 and EMD-1974). and regulating viral RNA synthesis (8). 1To whom correspondence should be addressed. E-mail: sarah.butcher@helsinki.fi. Ultrastructural studies on paramyxoviruses using electron This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cryomicroscopy (cryo-EM) combined with image processing have 1073/pnas.1105770108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1105770108 PNAS | November 1, 2011 | vol. 108 | no. 44 | 18085–18090 Downloaded by guest on September 27, 2021 materials for purification (sucrose versus the isotonic OptiPrep), had clear vesicles inside (Fig. 2C, Fig. S1A, and Movie S3). It has been postulated that the glycoproteins on the virion surface would interact with a layer of M protein adjacent to the inner leaflet of the viral membrane. The tomograms showed a membrane with apparently randomly distributed glycoproteins on the surface (Fig. 2, Fig. S1A, Fig. S2, and Movies S1, S2, S3, S4, and S5). We analyzed the density distribution across the membrane to investigate whether or not there was significant protein density directly underneath the membrane for five particles of similar size containing nucleocapsids (Fig. 3, and SI Fig. 1. Schematic diagram illustrating two possible ultrastructural models Materials and Methods; see Image Processing). This analysis revealed strong density for the membrane and for the glyco- for MV. (A) M coats the viral membrane and the nucleocapsid is free in the ∼ interior. (B) M coats the helical nucleocapsid. Color key: nucleocapsid, protein ectodomains ( 12 nm in length) (Fig. 3B) but only very brown; M, light blue; membrane, red; H, dark blue; F, yellow. low density proximal to the inner surface of the membrane. The average thickness of the membrane was 7 nm, which is similar to the value described earlier for the thinnest regions in the Sendai vaccine strain. The WT virus had better budding efficiency under envelope and much less than the 12 nm reported for areas in the conditions used (88% for WT, 0.1% for Edmonston) and Sendai where a matrix layer is possibly lining the membrane (14). a 100-fold better specific infectivity after purification (WT 2 × 109 Thus, MV seems to lack an ordered, continuous M protein layer − − PFU·mg 1 vs. Edmonston 2 × 107 PFU·mg 1)(SI Materials and next to the membrane. The average picture does not rule out that small membrane patches are coated with M. Methods; see Virus Growth and Purification) and was thus used – Two types of tubular structures could be detected in both for most of the detailed image analysis (Fig. 2 A D and Movies Edmonston and WT virion preparations. The tubes were ∼20 S1, S2, S3, S4, and S5). Similar features were seen in tomograms and 30 nm in diameter (Fig. 2 and Movies S1, S2, S3, S4, S5, and of the Edmonston preparations (Fig. S1 and Movie S6). The S6). In a total of 88 WT particles observed, 30-nm tubes could be reason for the difference in budding efficiency is not clear; it visually detected in 19 particles and 20-nm tubes in 12 particles, could be because of the cell types used or the differences in the both usually present in the same particle. The 20-nm tubes have genomes of the two strains, although the matrix and nucleo- the typical appearance of a MV herringbone-like ribonucleo- protein sequences are very similar. The WT particles varied in capsid. The 30-nm tubes also contain the same 20-nm tube but diameter from about 50 to 510 nm and exhibited a multitude of have an additional surrounding layer of protein density. Occa- different shapes (Fig. 2 A–D and Movies S1, S2, S3, S4, and S5). sionally, a partially uncovered nucleocapsid could be seen free in the sample, most likely originating from a disrupted virion (Fig. Some of the particles, from both strains, using different gradient 2F). The 30-nm tubes appeared relatively straight and rigid compared with the bare nucleocapsids that were often curved. Both ends and sides of some tubes were found touching the viral envelope.

Layer Covering the Nucleocapsid Is Composed of the Matrix Protein. Based on earlier observations of the Sendai virus M protein’s ability to self-assemble into helices (27, 28) and the MV M protein to bind the nucleocapsid (6–9), we hypothesized that the outer layer in the 30-nm tube could be composed of the M protein. Therefore, we carried out immunosorbent EM of infected cell lysates using anti-M and anti-N–coated grids (Fig. 4). A constant time (30 min) was used to observe the EM sample grids and images were collected whenever tubular structures were recognized. The anti-N grids were relatively densely covered by structures assigned almost solely to the 20-nm tubes

Fig. 2. MV ultrastructure and nucleocapsid organization. (A–D) Tomo- graphic slices of MV showing the general morphology of the virions. In the virions, two types of tubular structures with 20-nm (white arrow) and 30-nm Fig. 3. Membrane density profile. (A) A slab of density is shown for a to- (black arrows) diameters can be identified. See corresponding Movies S1, S2, mographic reconstruction of one virion. Part of the membrane density de- S3, and S4.(C) A vesicle inside a virion is indicated with an asterisk. (E–G) fined for the analysis is indicated as a gray surface. Some surface normals Different types of nucleocapsid structures from broken virions are shown: used for extracting and orienting subvolumes are shown as sticks. (B) Plot of a 30-nm structure in E, a partially-covered 20-nm structure in F, and a com- density distribution calculated from the extracted subvolumes as a function pletely bare 20-nm structure in G. Tomographic slices are 7.7-nm thick. (Scale of distance from the center of the membrane. The extent of the membrane bar, 100 nm.) and glycoprotein layer (F/H) is indicated with bars.

18086 | www.pnas.org/cgi/doi/10.1073/pnas.1105770108 Liljeroos et al. Downloaded by guest on September 27, 2021 Fig. 4. Electron micrographs of MV-infected and m and n cotransfected cell lysates prepared by immunosorbent EM. (A and B) Three different tubular forms can be observed from infected cells: (A, anti-N grid) 20-nm nucleocapsids with two different packing modes marked with a black arrow [similar to intact recombinant nucleocapsids (22–24)] and a white arrow [similar to trypsin-treated recombinant nucleocapsids (22–24)] and (B, anti-M grid) 30-nm tubes where matrix covers the nucleocapsids. In C a hollow 30-nm tube from an m and n cotransfected cell lysate (anti-M grid) is shown together with a 20-nm nucleocapsid. (Scale bar, 50 nm.)

(Table 1). The structures were of varying length and clearly of two different forms, one resembling the untreated recombinant nucleocapsid and the other resembling the trypsin-treated recombinant nucleocapsid (Fig. 4A) that have been reported previously (22–24). Anti-M grids had both the 20-nm tubes and the 30-nm tubes, approximately one-third being of the 30-nm type (Table 1). In these pull-down experiments, both the 20-nm and the 30-nm tubes were found as singular tubes and as aggregates of tubes reflecting M-to-M and M-to-N interactions, rather than cross-reactivity of the antibodies. Anti-P grids were used as controls to rule out the possibility that the P protein could form the outer helix, as it is known that P also interacts with N (29–31). On anti-P grids, fewer than 10 tubular structures in total could be observed and they were all of the 20-nm type. Hence, the 30-nm diameter tubes are matrix-covered nucleocapsids (MCNC). When a similar immunosorbent-EM experiment was conducted on lysates of m and n cotransfected HEK293E cells, no M-covered nucleocapsids were observed. Instead, 20-nm nucle- Fig. 5. Averaged structure of the MCNC. (A–D) Isosurface representations ocapsids of both types were prevalent on the anti-N grids, along of the MCNC structure. The structure is seen from the side in A to C,and with some hollow 30-nm matrix tubes on the anti-M grids (Fig. a slice taken along the axis is shown in D. Both the outer (blue) and inner 4C). This finding indicates that M is capable of assembling into (orange) parts show a clear helical twist. The transparent surfaces were helical structures without a nucleocapsid scaffold, as shown for rendered at a low threshold and the opaque surfaces at a high threshold (0.5 the Sendai and RSV matrix (27, 28, 32), and that some addi- σ and 1.0 σ above the mean density, respectively). (Scale bar, 10 nm.) The tional viral factor not present in cotransfected cell lysates is stars in D represent the five-start helical arrangement. (E) Translational self- correlation plot shows the correlation coefficient plotted as a function of needed for MCNC assembly. MICROBIOLOGY shift along the helical axis. (F) Rotational self-correlation plot shows the fi Matrix Layer Has a Helical Symmetry, Which Is Different from the correlation coef cient plotted as a function of rotation around the helical axis (solid line). The same function was plotted for a map correlated against Helical Symmetry of the Nucleocapsid. To analyze the structure of its copy, which had been rotated 180° to turn it upside down (dotted line). the MCNC, we carried out subvolume alignment and averaging The coloring in E and F corresponds to that in A to D. of MCNC segments extracted from the tomograms. The inner nucleocapsid and outer matrix layer were treated separately from fi fi one another in the re nement, as initial attempts to re ne both (Fig. 5E). This value is in line with previously reported values layers simultaneously were unsuccessful (SI Materials and Meth- (5.0–6.6 nm) for recombinant nucleocapsids (22, 24). The outer ods; see Image Processing). The averaged structure revealed two M protein layer is also a left-handed helix, but has a larger pitch concentric helices, indicating that the matrix layer also has he- of 7.2 nm (Fig. 5E). This value is identical to the one reported for lical symmetry (Fig. 5). Strikingly, this symmetry is different from purified Sendai M helices (27). Second, rotational self-correla- the symmetry of the helical nucleocapsid: First, the inner nu- tion plots clearly demonstrated that the nucleocapsid is a one- cleocapsid is a left-handed helix with a pitch (i.e., ridge-to-ridge start helix but the M protein layer is a five-start helix (Fig. 5F). distance) of 6.4 nm, as determined by autocorrelation analysis Third, the almost perfect correlation of the M helix with its 180° rotated (around the axis perpendicular to the helical axis) copy indicated that the M helix lacks directionality: that is, it has a so- Table 1. Immunosorbent EM of lysates from virus-infected cells called “dyad” axis (Fig. 5F). This finding would further suggest Anti-M Anti-N that the repeating structural units of the M helix are dimers, which have their twofold symmetry axis perpendicular to the MCNC* 54 15 helical axis. Although the limited resolution in our recon- Bare nucleocapsids 167 1828 † struction (4.4 nm, based on Fourier shell correlation 0.5 crite- Percentage of MCNC 32.3 (2.11) 0.82 (1.79) rion) did not allow us to demarcate individual subunits, dimers of *All nucleocapsids with partial or complete outer layer coverage were in- M would be consistent with data on Sendai M protein (28, 33). In cluded. contrast, the nucleocapsid helix correlated poorly with its 180° †SD from three individual experiments. rotated copy and thus has clear directionality, as expected based

Liljeroos et al. PNAS | November 1, 2011 | vol. 108 | no. 44 | 18087 Downloaded by guest on September 27, 2021 on visual inspection of the tomograms (Fig. 2 and Movies S1, S2, S3, S4, and S5) and earlier reconstructions of MV recombinant nucleocapsids (22–24).

Matrix-Covered Nucleocapsids Form Tightly Packed Bundles Inside the Virions. We carried out two independent sets of template matching by correlating first the averaged matrix helix and then the averaged nucleocapsid helix against the tomograms to position them segment by segment back into their original context (Fig. 6). The 3D search found most of the M-covered nucleocapsids that had been defined manually, some additional ones, and several stretches of bare nucleocapsids. The directionality of the nucleocapsid segments was in agreement with the directionality of the other segments in the same tube. The template matches to the M helices were often found pointing in two op- posing directions within the same tube, consistent with the fact that the M helix lacked directionality. Detection of the MCNC in the tomograms allowed us to study if the MCNC pack randomly or in a specific way. The MCNC were often detected in tightly-packed bundles. The average center-to-center distance in 20 pairs of nearly parallel MCNC was ∼30 nm, similar to the diameter of the MCNC, suggesting very close interactions within the bundles. The adjacent MCNC within the bundles were often antiparallel (Fig. 6 E–H). The number of MCNC per virion in the eight virions analyzed varied from 3 to 17 and the length of individual tubes from 46 to 160 nm. The total length of the detected MCNC in a virion varied from 230 to 1,400 nm (Table 2). Discussion The morphology of MV and other paramyxoviruses has been characterized extensively in the past using negative-stain EM. Except for the observations of filamentous nucleocapsids in these viruses, the interior of the virion has remained largely unchar- acterized because of limitations of the negative-stain technique. These limitations are overcome by cryo-EM, which allows 3D structural investigations of samples in their native hydrated state. A recent cryo-EM study showed that Sendai virions are highly pleomorphic (14), agreeing with the results of the present study. The MV membrane contains an apparently random distribution of surface glycoproteins that were so densely packed that we could not discern if, for example, H and F proteins were bound to each other on the surface, as biochemical data suggest (Fig. 2 A–D and Movies S1, S2, S3, S4, and S5), or partition in to different areas of the membrane based on curvature, as has been reported in influenza virus (34–38). Some of the MV particles analyzed in this study contained vesicles, similarly to those reported in Sendai virus (14). Thus, such vesicles may be a common feature of the paramyxoviruses. Our cryo-EM data give insight on the MV M protein organization. The M protein has been generally thought to line the inner leaflet of the membrane in budded virions, interacting with the viral glycoprotein tails (Fig. 1A). This hypothesis has been based on the observations that the M protein is translocated to cell membranes, has a tendency to bind membranes, and is capable of driving virus-like particle formation when expressed without other viral proteins (5, 39). However, an alternative hypothesis presented in Fig. 1B, driven by the data we present in this article, and still explaining these observations, suggests that the majority of the M protein in virions is in fact not lining the viral membrane, but rather forms a layer covering the nucleocapsids in a helical fashion. Bundles of the MCNC are then found inside the virions. This organization in turn casts doubt on Fig. 6. Organization of the MCNC in virions. (A) The averaged structures for the matrix (blue) and nucleocapsid (orange) filaments were placed back into models for MV assembly that assume M interacts with N only at the density map of a virion (one section is shown in gray scale, positive density the cytoplasmic membrane during budding. Several observations is black) (Movie S5). (B–D) End-on views of the bundles in A are shown from support the hypothesis that the MCNC forms already in the the directions indicated with arrows in A. One M-helix was hollow in C and one cytosol before transport to the cytoplasmic membrane: First, NC helix was bare in D,reflecting either inaccuracies in the computational early electron microscopy data indicate that there are both analysis or biological variation in the MCNC structure. (E–H)Differentexamples thinner “smooth filaments” and wider “granular filaments” in the of MCNC packing in virions are shown. The embedded schematic diagrams cytosol of infected cells (18, 19). The “granular filaments” illustrate possible connectivity between the MCNC filaments, consistent with probably correspond to the MCNC and concentrate toward the an antiparallel arrangement of neighboring filaments.

18088 | www.pnas.org/cgi/doi/10.1073/pnas.1105770108 Liljeroos et al. Downloaded by guest on September 27, 2021 Table 2. Number and length of MCNC in virions virus it is found next to the membrane as a layer, but after ex- Average length of MCNC* Total length posure to the low pH of the endosome it dissociates from the Virion No. of MCNC (nm) (nm) membrane (38). How does the matrix layer assemble on the nucleocapsids? 1 3 84 (11) 250 Image processing of the MCNC structure revealed first, that 2 3 77 (10) 230 native nucleocapsids, containing viral RNA in the presence of 3 4 97 (20) 390 additional proteins, such as M, L, and P, are similar in pitch to 4 5 120 (19) 580 those reported from untrypsinized recombinant nucleocapsids 5 10 72 (16) 650 (22, 24), indicating that no major structural changes occur in the 6 11 90 (31) 900 nucleocapsid upon matrix binding. Our data indicate that where 7 13 120 (30) 1,400 M assembles as a helix on to the outside of the nucleocapsid, the 8 17 74 (8) 1,200 resulting MCNC is rather straight, but flexible bare regions allow bundling of the nucleocapsid (Fig. 6 and Movie S5). Second, *SD in parentheses. there is an unexpected symmetry mismatch between the outer matrix helix and the inner nucleocapsid helix. This finding suggests some flexibility is required in the interaction between these cell periphery, close to where particles are observed to bud (18, proteins. There is abundant evidence from sequence predictions, 19). Second, in our immunosorbent EM analysis, MCNC could protease digestion, NMR, and CD spectroscopy, that the in- be detected in cell lysates of infected cells. Third, efficient nu- trinsically disordered NTAIL is exposed on the outside of assem- cleocapsid transport to the plasma membrane requires accu- bled nucleocapsids (22, 23, 43) and folds cooperatively on mulation of matrix in intracellular membranes (6). Taking these interaction with P (44). Additionally, interaction and mutational observations together, we suggest that the formation of the viral studies have shown that the N residues L523 and L524 are RNA-containing nucleocapsid, covered with M protein, is re- TAIL fi critical for binding M (8). Thus, we propose that the ribonucleo- quired for ef cient transport to the budding sites. The assembly capsid could help in nucleating the assembly of the M helix, but of the MCNC could also explain mechanistically how the M M–M interactions promote the further growth of the M helix and protein can act as a repressor of transcription and viral RNA define its organization. Supporting the hypothesis that M can self- synthesis (8, 9), as the matrix coat could prevent the polymerase assemble, we observed single-shelled, 30-nm diameter helices from binding to the viral RNA, wound on the outer edge of the similar to the MCNC outer layer in both immunosorbent EM of nucleocapsid (24). This repression could also be important transfected cell lysate (Fig. 4C)andinaviriontomogram(Fig. S2). during entry where M uncoating would be required for tran- How is the formation of segmental MCNC promoted and scription to start. controlled? Some possibilities that could be tested with an in vitro The MCNC were often found in tightly packed bundles, sug- assembly system are: (i) the tertiary structure of the RNA or ac- gesting that the M protein in different MCNC could interact with tive transcription causes flaws or bends in the ribonucleocapsid; each other. As the length of individual MCNC were too short to or (ii) other viral proteins, such as P, prevent M assembly (44). accommodate the whole genome, and the bundles seem to In conclusion, we have characterized the ultrastructure of MV contain antiparallel segments, it is likely that the individual and described a previously undescribed matrix-nucleocapsid MCNC in one bundle are linked together by bare regions of complex existing in the virions. We suggest a detailed model for nucleocapsid, difficult to resolve in the tomograms because of the organization of the matrix protein inside the virions in which various artifacts, such as the missing wedge. Supporting this M and nucleocapsid form a bundled two-layer helical structure finding, both bare nucleocapsids and MCNC were observed in for efficient packaging of the genome in the virions. Although the same virion. In addition, the total length of MCNC in any the MCNC may not be a common feature of the paramyxo- individual bundle was smaller than that expected for a full-length viruses, matrix proteins from other paramyxoviruses, and the genome (around 1 μm), so different bundles need to be con- more distantly related vesicular stomatitis virus (42) and influ- nected. Some of the particles could be polyploid (Table 2, viruses enza virus (36), also have a strong tendency to form helical 7 and 8) as described earlier (40). Because of the nature of structures, which reflects the protein’s propensity to self-assem- transmission cryo-EM, very large particles, (greater than ∼600 ble (27, 28, 32). Our revised model of measles virus, founded on MICROBIOLOGY nm in diameter and likely to be polyploid), through which the direct tomographic evidence, will direct future studies to inve- electron beam does not penetrate, were excluded from our stigate the importance of matrix assembly on to the ribonu- detailed analysis. cleoprotein in paramyxovirus budding and cell entry. M may have a role in compact packing of the genome, effec- tively reducing the total surface area of the virion, thereby aiding Materials and Methods budding. If the majority of the M protein in virions is organized Virus Growth and Purification. WT virus (a gift from I. Davidkin, Helsinki, around the nucleocapsids before reaching the plasma membrane, Finland) was grown in Vero-SLAM cells (a gift from Y. Yanagi, Fukuoka, as our results suggest, the driving force in curving the cell mem- Japan) and Edmonston vaccine strain (ATCC VR 24) in B-Vero cells. The brane for budding seems unlikely to be exerted by the multi- viruses were purified using ultracentrifugation in Optiprep and sucrose merization of the M protein on the inner leaflet of the plasma gradients (SI Materials and Methods;seeVirus Growth and Purification). membrane but is more likely to be driven by actin polymerization, as suggested by Bohn et al. (41). It is also possible that the M Cloning of m and n and Expression in HEK293E Cells. Full-length m and n were protein has at least two roles, one responsible for budding, the amplified by RT-PCR from TRIzol extracted MV WT RNA and cloned into other for recruiting the nucleocapsid to the budding sites. A plasmid pTT5SH8Q2 (a gift from Y. Durocher, Montreal, Canada) using NotI postranslational modification responsible for M separation into and HindIII restriction sites. HEK293E (45) cells were transfected with poly- two bands on SDS/PAGE gels (39) would be the most likely ethyleneimine and a 1:1 molar ratio of m- and n-containing vectors. Cells were harvested 3 d after transfection and lysed in 50 mM Tris-HCl pH 8.0, 150 nominator of these two roles. In contrast, Sendai virus particles do mM NaCl, 1% Nonidet P-40, and 0.5 mM Pefabloc SC (Sigma-Aldrich) pro- have matrix lining the envelope, so the budding and entry of these tease inhibitor and transferred to –80 °C until use. Expression of both pro- two viruses may well have diverged (14). Indeed, it has been shown teins was verified with Western blots using anti-M (MAB8910; Millipore) and that Sendai matrix does not control the transcription and repli- anti-N (2F3, Santa Cruz Biotechnology) antibodies. cation of the genome, as expected if matrix and nuclocapsid interact only at the plasma membrane (42). Interestingly, Sendai M Cryo-EM. Samples of purified virus were mixed with 10-nm colloidal gold and protein can assemble into helices on its own, and has been shown vitrified on holey carbon-coated grids (C-flat 200 or 400 mesh; Electron to coat nucleocapsids in preparations isolated from virions (28). In Microscopy Sciences). Cryo-EM was conducted at liquid nitrogen temperature influenza virus, the position of the matrix is dynamic. In budded and low-dose conditions using a 200-kV transmission electron microscope

Liljeroos et al. PNAS | November 1, 2011 | vol. 108 | no. 44 | 18089 Downloaded by guest on September 27, 2021 (F20; FEI). Images were recorded with a CCD camera (Ultrascan 4000; Gatan). grids were then blocked with 3% BSA in PBS for 60 to 90 min, washed with In total, 27 tilt series (typically ±60° at 2° increments) were collected in PBS, and incubated for 40 min on cell lysate drops from virus-infected or SerialEM (46) at underfocii ranging from 3 to 6 μm and at a nominal mag- plasmid-transfected cells. The grids were then washed and negatively stained fi × ni cation of 39,400 , resulting in a sampling of 0.38 nm per pixel. with 1% potassium phosphotungstate (pH 7.0). All steps were carried out at room temperature. The stained grids were observed with an F20 electron Image Processing. Image processing details are described in SI Materials and microscope at 68,000× magnification. Methods;seeImage Processing. Briefly, from tomographic reconstructions, individual MCNC segments were extracted as subvolumes that were then ACKNOWLEDGMENTS. We thank Ritva Kajander and Pasi Laurinmäki for subjected to subvolume alignment and averaging using Jsubtomo (47). The excellent technical assistance; Jyrki Hokkanen for graphics; the Biocenter fi inner and outer helices were re ned separately. The density distribution Finland National Cryo-Electron Microscopy Unit, Institute of Biotechnology, across the membrane was calculated from a subvolume average obtained Helsinki University, and the CSC-IT Center for Science Ltd. for providing from tomograms of the virus surface. facilities; Dr. Ilkka Julkunen and Dr. Irja Davidkin for wild-type virus and facilities; Dr. Yves Durocher for plasmid pTT5SH8Q2; Prof. Yusuke Yanagi Immunosorbent EM. Infected cells were lysed in PBS with 5% Triton X-100 and for the Vero-SLAM cell line; and Prof. Timo Hyypiä for useful discussions and comments on the manuscript. This work was supported by the Academy 1 mM Pefabloc SC to release subviral complexes. Transfected cell lysates were of Finland Centre of Excellence Programme in Virus Research 2006–2011, supplemented with 5% Triton X-100 to enhance solubilisation of M. For Grant 129684 (to S.J.B.); Academy of Finland Grants 130750 (to J.T.H.), immunosorbent EM analysis of the released complexes, carbon-coated 128539 (to P.S.), and 139178 (to S.J.B.); the Sigrid Juselius Foundation (S.J.B.); copper grids (Electron Microscopy Sciences) were glow-discharged and coated and Viikki Graduate School in Molecular Biosciences and European Molecu- with anti-M, anti-N or anti-P (9H4; Santa Cruz Biotechnology) for 10 min. The lar Biology Organization Short-Term Fellowship ASTF 171-2009 (to L.L.).

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