Structural rearrangement of infecting Sindbis virions at the cell surface: mapping of newly accessible epitopes

Sindbis virus glycoproteins E1 and E2 undergo a conformational alteration during early virus-cell interaction at the cell surface (D. Flynn, W. J. Meyer, J. M. MacKenzie, Jr., and R. E. Johnston, J. Virol. 64:3643-3653, 1990). Certain epitopes normally internal on native virus become accessible to monoclonal antibody (MAb) binding after attachment but before internalization of virus particles. These newly exposed epitopes, termed transitional epitopes, may be part of functionally important domains made accessible at the surface of the altered virus to facilitate entry into cells. Heating Sindbis virions at 51 degrees C for a short time induced a similar, although not identical, exposition of transitional epitopes on the E1 and E2 glycoproteins (W. J. Meyer, S. Gidwitz, V. K. Ayers, R. J. Schoepp, and R. E. Johnston, J. Virol. 66:3504-3513, 1992). In the current report, we have identified several of the transitional epitopes that become exposed as a consequence of early virus-cell interactions. Transitional epitope MAbs that bound to rearranged, heated virions and virus-cell complexes were used in antibody competition binding assays on heated Sindbis virions to map the spatial relationships between native, external, neutralizing antigenic sites and newly exposed transitional epitopes. Because the heated, rearranged particles retained their infectivity, MAbs that bound to transitional epitopes also were used to isolate MAb neutralization escape mutants. Sequencing the glycoprotein genes of the escape mutants identified specific E1 and E2 loci where mutation prevented MAb binding to transitional epitopes. One of the transitional epitopes identified (E2 residues 200 to 202) lies in the E2 190-216 region, which harbors two major neutralization sites, E2a and E2b, and an N-linked glycosylation site at E2 196. The glycosylation signal was eliminated by site-directed mutagenesis of a full-length cDNA clone of the Sindbis virus genome. The absence of a carbohydrate moiety did not expose the transitional epitopes mapped to this locus, suggesting that on native virions, the inaccessibility of the E2 200-202 determinant was inherent in the structure of the glycoprotein spike.