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

Abstract

Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix-matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices.

Fig. 1.

Schematic diagram illustrating two possible ultrastructural models 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, brown; M, light blue; membrane, red; H, dark blue; F, yellow.

Fig. 2.

MV ultrastructure and nucleocapsid organization. (A–D) Tomographic 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 (black arrows) diameters can be identified. See corresponding Movies S1, S2, S3, and S4. (C) A vesicle inside a virion is indicated with an asterisk. (E–G) Different types of nucleocapsid structures from broken virions are shown: a 30-nm structure in E, a partially-covered 20-nm structure in F, and a completely bare 20-nm structure in G. Tomographic slices are 7.7-nm thick. (Scale bar, 100 nm.)

Fig. 3.

Membrane density profile. (A) A slab of density is shown for a tomographic reconstruction of one virion. Part of the membrane density defined for the analysis is indicated as a gray surface. Some surface normals used for extracting and orienting subvolumes are shown as sticks. (B) Plot of density distribution calculated from the extracted subvolumes as a function of distance from the center of the membrane. The extent of the membrane and glycoprotein layer (F/H) is indicated with bars.

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 (–24)] and a white arrow [similar to trypsin-treated recombinant nucleocapsids (–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.)

Fig. 5.

Averaged structure of the MCNC. (A–D) Isosurface representations of the MCNC structure. The structure is seen from the side in A to C, and a slice taken along the axis is shown in D. Both the outer (blue) and inner (orange) parts show a clear helical twist. The transparent surfaces were rendered at a low threshold and the opaque surfaces at a high threshold (0.5 σ and 1.0 σ above the mean density, respectively). (Scale bar, 10 nm.) The stars in D represent the five-start helical arrangement. (E) Translational self-correlation plot shows the correlation coefficient plotted as a function of shift along the helical axis. (F) Rotational self-correlation plot shows the correlation coefficient plotted as a function of rotation around the helical axis (solid line). The same function was plotted for a map correlated against its copy, which had been rotated 180° to turn it upside down (dotted line). The coloring in E and F corresponds to that in A to D.

Fig. 6.

Organization of the MCNC in virions. (A) The averaged structures for the matrix (blue) and nucleocapsid (orange) filaments were placed back into the density map of a virion (one section is shown in gray scale, positive density is black) (Movie S5). (B–D) End-on views of the bundles in A are shown from the directions indicated with arrows in A. One M-helix was hollow in C and one NC helix was bare in D, reflecting either inaccuracies in the computational analysis or biological variation in the MCNC structure. (E–H) Different examples of MCNC packing in virions are shown. The embedded schematic diagrams illustrate possible connectivity between the MCNC filaments, consistent with an antiparallel arrangement of neighboring filaments.