Assembly, maturation and three-dimensional helical structure of the teratogenic rubella virus

Abstract

Viral infections during pregnancy are a significant cause of infant morbidity and mortality. Of these, rubella virus infection is a well-substantiated example that leads to miscarriages or severe fetal defects. However, structural information about the rubella virus has been lacking due to the pleomorphic nature of the virions. Here we report a helical structure of rubella virions using cryo-electron tomography. Sub-tomogram averaging of the surface spikes established the relative positions of the viral glycoproteins, which differed from the earlier icosahedral models of the virus. Tomographic analyses of in vitro assembled nucleocapsids and virions provide a template for viral assembly. Comparisons of immature and mature virions show large rearrangements in the glycoproteins that may be essential for forming the infectious virions. These results present the first known example of a helical membrane-enveloped virus, while also providing a structural basis for its assembly and maturation pathway.

Fig 1. Rubella virion morphology.

(A) Section from a rubella virus tomogram showing the different morphologies of rubella virions. Scale bar corresponds to a length of 200 Å. (B) Cross-section of a rubella virion. The dashed rectangles in red show individual rubella glycoprotein spikes. The dashed ovals in red indicate thin densities that connect the inner nucleocapsid shell to the outer glycoprotein plus membrane shell. (C) Surface of a rubella virion showing the glycoprotein rows. Black arrows mark the direction of the rows. Scale bar in panels B and C correspond to a length of 100 Å. Black is high density in all panels.

Fig 2. Helical organization of the rubella virus glycoproteins.

Representation of three different rubella virions (A, B and C) showing the organization of their surface glycoprotein rows. The virions have been extracted and rendered using UCSF Chimera [61] without any averaging procedures (Materials and methods). The extracted virions have been low pass filtered to 75 Å and hence, the surface glycoprotein rows appear as elevated ridges on the outer membrane surface. Scale bar is 100 Å in length. The surface contour is chosen at 0.81 standard deviations above average. The pitch of the helix in Fig 2A–2C is 533 Å, 390 Å and 0 Å, respectively. Further analysis of the glycoprotein rows using sub-tomogram averaging is shown in S1 Fig.

Fig 3. Structure of rubella virus glycoprotein spikes.

(A) Sub-tomogram averaged structure of the rubella glycoprotein spike (light blue) is shown placed on a membrane surface (yellow). The membrane surface has been modelled by extracting a lipid bilayer portion from a co-purified membrane vesicle in the un-averaged virus tomograms. The left and right panels are rotated 90° with respect to each other. (B) The same figure as in panel A, showing the rubella E1 ectodomain’s atomic structure fitted into the averaged density. The yellow star indicates the location of the rubella E2 ectodomain. The parenthesis in black indicate immunogenic surface regions on E1. Scale bars in panels A and B correspond to a length of 25 Å. Intermediates from the sub-tomogram averaging procedures are shown in S2 Fig. The Fourier Shell Correlation (FSC) curve calculated to estimate the resolution of the averaged glycoprotein spike map is shown in S3 Fig. See also S1 Table. (C) Cross-section of a rubella virion showing a representative glycoprotein row. Left panel shows the original tomogram section. The right panel shows the same section after placing the averaged glycoprotein spike (blue) (8X binned) into the tomogram. (D) Cross-sections showing a top view of the same glycoprotein row as in panel C. Black arrow indicates the glycoprotein row being considered. In panels C and D, scale bar is 50 Å long and black represents high density.

Fig 4. Nucleocapsid organization in rubella virions.

(A) Cross-section at the nucleocapsid surface of a rubella virion tomogram, showing a grid-like pattern of the nucleocapsid units (dashed red box). Scale bar is 50 Å long. (B) and (C) Left panel shows a tomogram section at the surface of the rubella virions; the right panel shows a section at the nucleocapsid surface. Red arrows indicate the glycoprotein rows and the corresponding nucleocapsid rows. Scale bar corresponds to a length of 100 Å. Black represents high density. A ball and stick model for the glycoprotein and nucleocapsid organization is given in S4 Fig.

Fig 5. In vitro assembled nucleocapsid cores.

(A) Negative stain image of purified rubella virus nucleocapsid cores. Black arrows indicate the co-purified capsid tetrameric units. Scale bar is 500 Å long. Agarose gel analysis of the effect of nuclease on the nucleocapsid cores is shown in S5 Fig. (B) Negative stain images of linear assemblies of capsid tetramers. Scale bar is 100 Å long. In panels A and B, white is high density. (C) and (D) Left panels show tomogram cross-sections from nucleocapsid core particles. Right panels show the end-on view of the nucleocapsid core surface that are indicated in the left panels by a dashed black line. Scale bars in panel C and D are 50 Å and 100 Å long, respectively. Black represents high density. (E) Side view of the sub-tomogram averaged density of the recombinantly produced nucleocapsid tetramers. (F) A single capsid unit density showing the fitted C-terminal domain of the capsid protein. Left panel shows the side view whereas the right panel shows the top view. Scale bar is 25 Å long. A more detailed fitting result for panel F is given in S6 Fig. See also S1 Table.

Fig 6. Cryo-electron microscopy image of purified immature rubella virions.

(A) The approximately 500 Å diameter, uniformly dense and smooth particles in the images represent the immature virions captured on a 1k×1k CCD camera with a dose of 20 e^-/Ų. (B and C) Zero degree tilt images of mature rubella virions collected using a 2k×2k CCD camera (dose: 2e^-/Ų) and with a Gatan K2 Summit direct electron detector (dose: 0.8e^-/Ų) respectively. The spiky nature of the mature rubella virions (indicated by black arrow heads) is seen even under the very low dose images in panels B and C. All panels also contain 100 Å BSA-gold particles (small and very dense particles). Scale bar is 500 Å long. Black represents high density.