Locations of carbohydrate sites on alphavirus glycoproteins show that E1 forms an icosahedral scaffold

Abstract

There are 80 spikes on the surface of Sindbis virus arranged as an icosahedral surface lattice. Each spike consists of three copies of each of the glycoproteins E1 and E2. There are two glycosylation sites on E1 and two on E2. These four sites have been located by removal of the glycosylation recognition motifs using site-specific mutagenesis, followed by cryoelectron microscopy. The positions of these sites have demonstrated that E2 forms the protruding spikes and that E1 must be long and narrow, lying flat on the viral surface, forming an icosahedral scaffold analogous to the arrangement of the E glycoprotein in flaviviruses. This arrangement of E1 leads to both dimeric and trimeric intermolecular contacts, consistent with the observed structural changes that occur on fusion with host cell membranes, suggesting a similar fusion mechanism for alpha- and flaviviruses.

Figure 1. CryoEM Images of the Wild-type Reconstructions Sindbis Virus

(a) Depth queued representation overlaid with a T-4 lattice, (b) surface shaded exterior, and (c) cross section. Shown is the icosahedral surface lattice and the chosen icosahedral asymmetric unit. The coordinate system used to define the position of the carbohydrate sites (Table 3) is also indicated. The membrane is seen clearly in (c) and is situated between 205 and 245Å radii.

Figure 2. Radial Sections Showing the Native Virus Densities (Top) and Difference Map Densities (Bottom) at the Radii Corresponding to the Different Carbohydrate Sites

As T = 4 related peaks will be at roughly the same radius, these maps demonstrate the quasi-symmetry for each carbohydrate site. The maps are color coded to correspond to the colors in Figure 3. Shown also is the icosahedral lattice and the outline of the chosen icosahedral asymmetric unit. A 200Å scale bar is shown at the bottom right.

Figure 3. Composite of Difference Map Sections Normal to the Central Icosahedral Fold Axis for SINV (a, b, and c) and RRV (d, e, and f)

(a) E2-196 (blue contours) and E2-318 (magenta contours) mutation sites. (b and c) E1-139 (green contours) and E1-245 (red contours) mutation sites. (d) E1-141(green contours) and E2-200 (blue contours). (e) E1-141 (green contours) and E2-262 (pink contours). (f) E2-262 (pink contours) and wild-type density (black contours). Appropriately colored lines indicate with which spike each difference peak is associated in (a) through (e). The triangle delineates the icosahedral asymmetric unit (see Figure 1) projected down an icosahedral 2-fold axis on planes that are between 260 and 335Å from the center of the virus. Contours are at 2s intervals. The four quasi-equivalent peaks are marked A, B, C, and D for the T = 4 related mutation sites. Symmetry axes surrounding the icosahedral asymmetric unit and the identification of the i3 and q3 spikes are marked in (f).

Figure 4. Stereo View of Carbohydrate Sites (Shown as Difference Density) Associated with One Spike

The color code is as in Figure 3. Black lines between E1-139 and E1-245 and between E2-196 and E2-318 correspond to E1 and E2 monomers, respectively. The cryoEM density of the wild-type map is shown in light blue for the spike and in yellow for the phospholipid membrane.

Figure 5. The E1 Icosahedral Scaffold Consistent with the Carbohydrate Sites Corresponding to Figure 3

Shown also are the sites of the carbohydrate moieties at E1-139, E1-245, and E2-318 colored as described in Figure 3. The orthogonal projection for the whole top hemisphere of the virus (top left) is viewed down an icosahedral 2-fold axis. Details about one spike are shown in the right-hand and bottom panels, viewed down a quasi-3-fold axis and fitted into the slabbed electron density (grey) of the wild-type cryoEM reconstruction. The top panels show the E1 molecules as cylinders, 80Å long and 28Å diameter. The bottom panel shows a stereographic view of the E1 molecule C_a backbone tracing, assuming its homology with the TBEV E glycoprotein. The putative site of the fusion peptide (E1 79 to 97) is shown in red. Note that the latter assumption (as opposed to the cylindrical simplification) avoids steric conflict with the carbohydrate site associated with E2-318.