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

<div><p>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 <i>in vitro</i> 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.</p></div

Nucleocapsid organization in rubella virions.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s005" target="_blank">S4 Fig</a>.</p

Helical organization of the rubella virus glycoproteins.

<p>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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.ref061" target="_blank">61</a>] 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s002" target="_blank">S1 Fig</a>.</p

Structure of rubella virus glycoprotein spikes.

<p>(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 <i>A</i>, 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 <i>A</i> and <i>B</i> correspond to a length of 25 Å. Intermediates from the sub-tomogram averaging procedures are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s003" target="_blank">S2 Fig</a>. The Fourier Shell Correlation (FSC) curve calculated to estimate the resolution of the averaged glycoprotein spike map is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s004" target="_blank">S3 Fig</a>. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006377#ppat.1006377.s001" target="_blank">S1 Table</a>. (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.</p

Class averages of radial averaged particle images of the WNV/E16 Fab complex at pH 6.0.

<p>The classification suggests the presence of at least four different radial expansion intermediates that represent advancing stages of the E protein layer expansion (IM-1 through 4). The number of images grouped in each class is indicated in percent below the respective class average. Most of the particles clustered in expansion stage IM-4, in which the outer protein layer, presumably composed of E and Fab molecules, reaches its maximum radius separated by an about 60 Å-wide gap from the outer lipid leaflet. A representative class average of the complex at pH 8 is shown on the left. The red lines indicate the radial limits of the nucleocapsid core (NC), the lipid bilayer (LB), the E protein layer (E) and the Fab molecules (Fab) in the pH 8 structure. Areas of high density are depicted with black pixels, low density areas are shown in white.</p

CryoEM images of WNV, alone or in complex with E16 Fab, in different pH environments.

<p>(A) WNV/Fab complex at pH 8. (B) WNV/Fab complex at pH 6. Acidification triggered an expansion of the virus particles that resulted in a halo-effect around the dense nucleocapsid core. Inset: Examples of back-neutralized particles showing the irreversibility of the low-pH induced changes. (C) WNV at pH 8. (D) WNV at pH 6. Strong particle aggregation was detected when WNV was exposed to low pH in the absence of E16. Acid-induced structural changes similar to (B) were observed for some virions at the edge of the aggregates, as indicated by the arrow. The scale bars represent 500 Å.</p

Equatorial slices of cryoEM image reconstructions of WNV in complex with E16 antibody fragments in different pH environments.

<p>(A) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 8, rendered at 23 Å resolution. Red arcs (1 through 5) specify the outer radii of the nucleocapsid core (154 Å), lipid bilayer (205 Å), E glycoprotein shell (247.5 Å), scFv molecules (278 Å), and Fab fragments (318.5 Å) from the viral center. The positions of the icosahedral two-, three- and fivefold axes are indicated with black arrows and numbers. (B) WNV/Fab complex (top half) and WNV/scFv complex (bottom half) at pH 6, rendered to 25 Å resolution. The red arcs (6 and 7) specify the outer radius of the expanded E/scFv (317.5 Å) and E/Fab (347 Å) protein layer, respectively. The low pH triggered radial expansion of the E/scFv or E/Fab protein shell resulted in a ∼60 Å wide shell of low density between the lipid bilayer and the expanded outer protein layer, as indicated by the green arrow. The scale bars represent 100 Å.</p

CryoEM image reconstruction of WNV in complex with E16 antibody fragments at pH 8.

<p>(A) Stereoscopic view of a surface rendering of WNV (green) complexed with E16 Fab (blue) at 23 Å resolution <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000672#ppat.1000672-Kaufmann1" target="_blank">[14]</a>, viewed down an icosahedral twofold axis. The black triangular outline identifies an icosahedral asymmetric unit. (B) Same as A, but depicting the complex of WNV and E16 scFv. E16 scFv binds WNV in a similar fashion as E16 Fab. Only two of three epitopes in the asymmetric unit are utilized. The fivefold proximal epitope is occluded because of steric hindrance.</p

Model of the initial stages of the pH-triggered E rearrangement on the viral surface during flavivirus cell entry.

<p>Domains DI, DII, and DIII of E are colored red, yellow, and blue, respectively. The partially alpha-helical stem region is shown in magenta and the transmembrane helices in grey. The fusion loop at the distal end of DII is indicated in green. Low endosomal pH weakens inter- and intramolecular E contacts and induces the outward extension of the whole stem region or its N-terminal portion including helix H1, followed by the dissociation of the E dimers. Further structural repositioning of the E monomers allows the interaction with the endosomal target membrane via the fusion loop. E16 was shown to inhibit the formation of fusion-active E trimers, most probably by interfering with the rearrangement of the E domains as a consequence of adding mass to DIII and sterically clashing with neighboring E and Fab molecules. However, E16 may also sterically prohibit the contact of E with the target membranes.</p

CryoEM data overview for the WNV/Fab E16 and the WNV/E16 scFv complexes at pH 6.

a<p>The cryoEM micrographs were digitized at 7-µm and 6.35-µm intervals, respectively, using a Zeiss SCAI or Nikon 9000 scanner. Sets of four pixels were subsequently averaged.</p