Promotion of virus assembly and organization by the measles virus matrix protein

Abstract

Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.

Fig. 1

Cryo-ET of MeV-infected HeLa cell. a An intermediate magnification image montage of an Edm MeV-infected HeLa cell. Site of MeV assembly is indicated by dashed white box and is the location where the tilt series was acquired. Asterisks indicate released MeV particles, white arrows indicate MeV assembly site, and red dashed lines indicate the cell membrane. b Tomographic slice (1.18 nm thick) of Edm MeV assembly site. Black arrows indicate areas where M is absent on the viral membrane (b, c). c 3D segmentation view of the cryo-ET data (b). Glycoproteins (purple), viral membrane (red), M (green), RNP (gold), and actin filaments (magenta) at the assembly site. d–f Sequential tomographic slices (1.18 nm thick) of a released virus particle (not shown in a) showing glycoprotein layer (d), M layer (e) and RNP layer (f). Arrowheads indicate the same regions at different Z heights. Scale bars are 500 nm (a) and 100 nm (b–f)

Fig. 2

MeV M protein forms a 2D paracrystalline array. a Tomographic slice showing a representative M array from Edm MeV-infected HeLa cells. Inset is the power spectrum indicating a four-fold symmetric structure with 7.8 nm spacing between subunits. b Sub-volume average of the M protein array indicates an improved SNR structure with regular spacing. Black is density. c Model fitting of the NDV M dimer into the averaged MeV M array density. Cyan and gold represent each monomer of the NDV M dimer. d The enlarged view of (c), with blue and gold arrowheads indicate the two dimer interfaces. Scale bars are 100 nm (a), 10 nm (b, c), and 5 nm (d)

Fig. 3

MeV F glycoprotein forms a 2D paracrystalline lattice with four-fold symmetry. a Tomographic slice showing the F glycoprotein layer from recMeV-(H-118∇41×)-infected HeLa cells at an assembly site. The inset is the power spectrum of the F layer, which indicates four-fold symmetry with subunit spacing of ~11 nm. Black is density. b The enlarged region (a) (white box) with an isosurface rendering showing the F ordering. Three F trimer densities were fitted using PIV5 pre-fusion F glycoprotein structure (PDB ID: 2B9B). c Sub-volume averaged structure of the F layer showing spacing with four-fold symmetry. Black is density. d Measurements between two nearest neighboring F glycoproteins (white line in b) indicate an average distance of ~11 nm, consistent with the power spectrum analysis and the sub-volume averaged structure (white line in c). The error bar represents the standard deviation (s.d.) of 100 measurements (graph data points). e, f Sub-volume average of F glycoprotein trimer indicates F is in the pre-fusion conformational state, validated by model fitting of PIV5 prefusion F structure (PDB ID: 2B9B). Scale bars are 50 nm (a) and 10 nm (b, c, e, f)

Fig. 4

Sub-volume average of the MeV F–M lattice layers. a-f Tomographic slices of the ordered F–M lattice. Pixel size is 5.88 Å at binning 2. Central slices at Z = 65 (black, M) and Z = 87 (blue, F) are shown for Edm (a–c) and recMeV-(H-118∇41×) (d-f). g-i Segmentation view of recMeV-(H-118∇41×) F–M lattice demonstrates the densities corresponding to M (h), F (i) and the F–M lattice overlay (g, F on top of M). Subunit distance between M (black line) is 11 nm and F (blue line) is 7.8 nm. Black is density in a–f. j Graphic representation of F and H in recMeV-(H-118∇41×) virus strain. Note the height differences in F and H in this virus strain. Black is density. Scale bars are 5 nm

Fig. 5

MeV RNP average and its spatial organization with M lattice. a Global average of MeV RNP with central slice side and top views (left), cartoon representation (middle), and isosurface rendering (right). b Classification reveals two classes with different helical pitches. c, d RNP class average distribution in the raw tomograms. White arrowheads indicate the directionality of the RNP. e, f The enlarged view of the white box in (d) showing tomographic slices of MeV at M layer (e) and RNP layer (f). Black squares indicate M lattice; red helices indicate the RNP helices. Note the directionality of M lattice is in register with RNP directionality. g Schematic model of M and RNP interaction and 3D organization. Black is density. Scale bars are 10 nm (a, b), 100 nm (c, d), 50 nm (e and f), and 20 nm (g)

Fig. 6

MeV assembly model. At the assembly site, viral components, including glycoproteins F and H, matrix protein (M), and RNP complexes, accumulate at the plasma membrane to initiate the assembly process. Actin filaments are potentially involved during the process. At the assembly site, M is the driving force which orchestrates the whole process by interacting with F and RNPs in a timely and spatially organized manner: M forms a 2D lattice under the envelope, F forms another 2D lattice interspersed above the M lattice, and RNP complexes lie under the M lattice with some degree of flexibility. H is incorporated at sites of assembly but does not appear with high spatial ordering. Once assembled, viral particles undergo an unknown scission event and bud off the plasma membrane. After release, the viral particles are free to infect the next cell