An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells vi a a membrane fusion reaction triggered by acidic pH in the endocytic pathwa y, Fusion is mediated by the spike protein El subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrim er during fusion, We have characterized four monoclonal antibodies (MAbs) s pecific for the acid conformation of El, These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptid e. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map th e binding site, we selected for virus mutants resistant to one of the MAbs, Ela-1, One virus isolate, SFV 4-2, showed reduced binding of three acid-sp ecific MAbs including Ela-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 muta nt was fully infectious, formed the F1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Dec reased binding of MAb Ela-1 was observed under a wide range of assay condit ions, strongly suggesting that the E1 G157R mutation directly affects the M Ab binding site. These data thus localize an E1 region that is normally hid den in the neutral pH structure and becomes exposed as part of the reorgani zation of the spike protein to its fusion-active conformation.