In general, the analysis of a number of strains of Sindbis virus has revealed amino acid differences of potential importance for virulence at relatively few positions in the E2-glycoprotein. Only 10 amino acid changes are potentially implicated, and 9 of these 10 lie in the N-terminal half of the protein (Fig. 1.). Currently, there is strong evidence to implicate 3 of these positions (E2-55, -114, and -172) in virulence (Table V). As more recombinant viruses are prepared and analyzed, the evidence for or against the relevance of other changes should become apparent. As is generally true in alphaviruses, the E1 gene is more invariant than E2 and analysis of several strains has revealed amino acid changes at only four positions (Fig. 2). Two, 72, and 75, are just N-terminal to the hydrophobic segment postulated to be the site of fusion activity, suggesting the possibility that virus entry into the host cell could be affected by amino acid differences at these locations. The other two changes (at 237 and 313) are distant from the fusion site on the linear molecule, but changes at 313 do affect the pH fusion suggesting participation of this site in providing stability to the glycoprotein trimers. The mechanism of altered virulence associated with any amino acid change in the E1- or E2-glycoproteins has yet to be determined. The change at E2-114 associated with reduced virulence in mice shows reduced latency and increased virulence in BHK-21 cells in vitro. This suggests that some changes result in enhanced replication that is host cell-specific. There are several points in the replication cycle of Sindbis virus where the glycoproteins and their ability to undergo conformational changes play an important role in efficiency of replication. These include attachment, fusion, transport through the Golgi, assembly, and budding from the cell surface. Some steps in replication involve host cell proteins (Baric et al., 1983), so that there may be unique, unexplored interactions with neurons or ependymal cells leading to increased neurovirulence for mice that are not represented in the typical BHK, Vero, or chick embryo fibroblast cell culture system. The task now will be to determine why specific amino changes in the proteins of Sindbis virus cause such dramatic changes in the biological properties of the virus.