Organization of the SINV polyprotein. Structural proteins are first translated as a polyprotein, which is then cleaved in a series of distinct processing steps to generate separate CP (blue), E3 (purple), E2 (green), 6K (orange), and E1 (red) proteins. The C-terminal two-thirds of CP adopts a protease domain structure. Each of the E1 and E2 glycoproteins has a large ectodomain containing two N-linked glycosylation sites (green arrowheads), a short stem region, a single TM helix (hatched regions), and a cytoplasmic domain. cdE1 and cdE2 consist of 5 (red characters) and 33 (green characters) residues, respectively. Interactions between cdE2 and CP occur in three regions each in CP (CP-RI, CP-RII, and CP-RIII) and cdE2 (cdE2-RI, cdE2-RII, and cdE2-RIII). Asterisks highlight key residues in the cdE2 that interact with CP as discussed in the text.

Overview of the SINV structure. (a) Shaded-surface representation of SINV cryo-reconstruction, color cued radially to emphasize the layered nature of the alphavirus structure (see below). The 80 prominent, trimeric spikes are positioned at threefold (black triangle) and quasi-threefold (white triangle) points in a T=4, icosahedral lattice (black lines). One of the 60 asymmetric units is highlighted by green shading, and thin black lines approximate the boundaries of individual subunits. (b) Same as (a) but with the front half of the reconstructed density map removed to reveal internal features of the SINV structure. The outer protein shell, composed solely of the E1 and E2 glycoproteins, is divided morphologically into spikes (blue) and the underlying skirt (magenta). The outer and inner leaflets of the viral membrane appear as two concentric, roughly spherical shells of density (cyan), crossed by distinct E1 and E2 TM helices (also in cyan). The ordered, protease domains of the CP (green) lie beneath the membrane and surround a layer of density (yellow) that is attributed primarily to the N-terminal segments of the CP. The remaining disorganized density (red) at the core of the virion is attributed primarily to the RNA genome. The scale bar represents 100 Å.

Atomic modeling of SINV cryo-reconstruction. (a) Stereo ribbon diagram representation of the E1–E2 heterodimer. Highlighted in the E1 molecule (red) are the two glycosylation sites (N139 and N245) and the fusion peptide (yellow; black arrow). Two models of the E2 structure (A and C domains) are shown: one found in crystals (cyan) and the other obtained after fitting it into the SINV cryoEM density map (green). The E2 B domain (blue), a homology model derived from the CHIKV E2 crystal structure (see Materials and Methods), is shown in its fitted position, where it lies directly above and covers the E1 fusion peptide. The single E2 glycosylation site (N196) in the E2-N318Q mutant is labeled. (b) Same as (a) for the final E1–E2 heterodimer model fitted into the SINV cryoEM density map (grey mesh). (c) Same as (b) but with a complete (E1–E2)₃ model fitted into the density map and viewed from above a threefold symmetric spike. The N196 glycosylation sites appear as clearly defined protuberances at the top of the spike (green spheres), the E1-N139 sites are at the far corners of the skirt (red sphere), and density at the E1-N245 sites (center of the spike) is not as well defined as for the other two sites. (d) Pseudo-atomic models of the E1 (red) and E2 (green) stem and TM helix regions, and the CP protease domain (blue) fitted into the SINV density map. At the chosen density threshold, the membrane bilayer shows up as a pair of thin layers that pucker inwards towards the virion center (bottom of panel). The stem regions (double arrow) are depicted in darker colors to distinguish them from the TM regions of the models.

SINV pentamer model. (a) Five copies of the CP protease atomic model fitted into the SINV density corresponding to the pentameric capsomer as viewed from the center of the virion. The ribbon model is color coded (blue– red rainbow) starting at CP residue 114. Strong intracapsomer subunit–subunit interactions are observed and include a potential salt bridge between R174 (blue sphere) and E236 (red sphere). (b) Same as (a) but including the layer of unmodeled density that lies immediately below the ordered protease domain layer and is ascribed primarily to the disordered, 113-residue, N-terminal region of CP. This layer appears as a network of density that supports both intra- and intercapsomer interactions. (c) Side view of the SINV map with density attributed to the CP protease domains subtracted away, to reveal a stretch of density leading from residue 114 (blue spheres) to the network below and modeled as eight residues ending at residue 106 (yellow sphere). The additional difference density at low radii in this view was interpreted as including most of the remaining residues of the CP N-terminus (amino acids 1–105). Left pair of arrows highlights the upper and lower bounds of the region of the map shown in (a), whereas the right pair highlights the region shown in (b).

TM helices and cdE1 and cdE2. (a) Stereo view of a slab of the SINV density map (gray mesh) that, for clarity, includes just the inner half of the membrane and the protease domain of the CP pentamer and viewed towards the center of the virion. The fitted E1 (red) and E2 (green) TM helix models, the cdE2 model (dark green), and CP (blue) are shown. (b) Close-up, side view in stereo of a small volume of the density map showing two pairs of TM helices, the cdE1 and cdE2 models, and the top portion of CP. Unassigned density at the top and center of the panel is attributed to the phospholipid head groups in the outer and inner leaflets of the bilayer, respectively. (c) Same as (b) showing a highly magnified view of a loop in CP (amino acids 248–252) that moves several angstroms from its position in the crystal structure (blue) to one that fits the density map (yellow). This movement opens the CP pocket and allows cdE2 to bind to it.

Interactions between cdE2 and CP. Stereo view shows the full-length cdE2 model (from K391 to A423) and the path it follows into and back out of the CP hydrophobic pocket. Residue W247 divides the pocket into upper and lower compartments, which contain F166 and Y180, respectively. The loop in cdE2 starting at R393 dives down into the pocket with Y400 and L402 making contacts with CP residues inside the upper and lower compartments of the pocket. Upon exiting the pocket, cdE2 forms a short helix that approaches the inner membrane and ends at C-terminal residue A423. The N-terminal residue in the CP crystal structure is R114.

One-step growth analysis of a subset of CP and cdE2 mutants. BHK-15 cells were infected at a multiplicity of infection of 2 with WT or mutant virus, and the supernatant was collected at hourly intervals. Each experiment was conducted in triplicate, and the virus released in the supernatants at 4, 6, 8, 10, and 12 h post-infection was determined by plaque assay. The mean value of each virus titer at each time point is plotted in the growth curves. For clarity, the labels for the growth curves are arranged from top to bottom to correlate with the amount of virus release at the 12-h post-infection time point.

Glycoprotein trimers cross-link groups of three capsomers in the NC. A stereo view down the pseudo-3-fold axis of the SINV virion shows how the three “legs” of an (E1–E2)₃ trimer (E1 in red and E2 in green) interconnect a pentamer and two hexamers (all in blue) in the underlying NC. Each leg consists of an E1–E2 TM pair and the corresponding cytoplasmic domains (cdE1 and cdE2).