Structural localization of the E3 glycoprotein in attenuated Sindbis virus mutants

Abstract

We have determined the three-dimensional structures of the wild-type Sindbis virus and two of its mutants that retain the E3 sequence within PE2. Using difference imaging between these mutants and the wild-type virus, we have assigned a location for the 64-amino-acid sequence corresponding to E3 in the mutant spike complex. In the wild-type virus, the spike is composed of an E1-E2 heterotrimer. The E3 protein was found to protrude midway between the center of the spike complex and the tips. Based on these results and the work of others, we propose a distribution for the functional domains of the spike proteins within the structure of wild-type Sindbis virus. Within the structure of the virus, the E1 domains form the central portion of the spike complex, while the tips are formed by the E2 domains that flare out from the center of the complex. The structural similarity between these Sindbis virus mutants and Ross River virus suggests that E3 may also be present in the latter, which is also a member of the Alphavirus genus.

FIG. 1

Protein composition of wild-type and noninfections PE2 mutant (PE^wt and PE^mut) Sindbis virus. Samples were ³⁵S radiolabeled and analyzed by electrophoresis on an SDS–10% polyacrylamide gel. Lane 1, wild-type Sindbis virus; lane 2, TRSB-NE2G216 (infectious PE2 mutant). Positions of the capsid protein, the two spike proteins E1 and E2, the PE2^mut, and a weak PE2^wt band are shown. The PE2^wt band is typical and is due to a small amount of uncleaved PE2 that escapes cleavage to E2. PE2^mut runs as a higher-molecular-weight band due to an extra glycosylated site. The E1 and E2 bands run as a smear in the wild-type lane because of their similar molecular weights (Table 1).

FIG. 2

Selected regions of 100-kV flood-beam electron cryomicrographs of TRSB (wt) (A), TRSB-N (noninfectious mutant) (B), and TRSB-NE2G216 (infectious mutant) (C). In the case of the infectious mutant, the magnification is lowered to display a larger field of the specimen area. The white arrows indicate collapsed virus envelopes, while the black arrows indicate free nucleocapsids. Black scale bars, 700 Å; white scale bar, 1,000 Å.

FIG. 3

Surface representation of three-dimensional reconstructions shown from a threefold view: TRSB (wt), TRSB-N (noninfectious mutant), and TRSB-NE2G216 (infectious mutant). The appropriate contour level for surface rendering was determined for the T=4 virus envelopes by assuming a protein density of 1.325 g/cm³ and by using the published molecular weights of the viral components compiled from the sources shown in Table 1.

FIG. 4

Superimposition of computationally isolated trimers from the wild type and the noninfectious PE2 mutant. The mutant trimer is displayed in wire frame with the blue density denoting 1.325 g/cm³ and the red indicating a denser region of the protein. The wild-type virus trimer is surface rendered in semitransparent yellow. We have assigned the major mutant protrusion that extends outside the wild-type trimer to the E3 region of the mutant PE2.

FIG. 5

Equatorial sections of the wild-type (A) and infectious mutant (B) structures shown along the twofold axis. The arrows indicate the densities present in the mutant which we have attributed to the E3 region of the PE2 mutant.

FIG. 6

Proposed model for the functional domains of the spike proteins and membrane fusion. (A) In the spike trimer, the E2 functional domain is the most outwardly exposed structural protein, while the E1 fusigenic domain is located in the center of the spike complex. E3 is on the outer edge of the E2 spike protein. (B) During attachment, the tripod-like complex forms a specific and stable three-point interaction with the host cell through interactions with cell receptors. These interactions induce a conformational change in the structure of the trimer in which the E2 tips are separated, bringing the center of the spike and the E1 homotrimer closer to the host membrane. As the trimer tips spread apart, conformational changes that expose the fusigenic peptides of E1 occur in the spike proteins. At the same time, the center of the spike containing the E1 trimer is brought close to the host membrane, where the fusigenic peptides of E1 are exposed and initiate fusion.