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.

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