Search

Semliki Forest virus serves as a paradigm for membrane fusion and assembly. Our icosahedral reconstruction combined 5276 particle images from 48 cryo-electron micrographs and determined the virion structure to 9 A resolution. The improved resolution of this map reveals an N-terminal arm linking capsid subunits and defines the spike-capsid interaction sites. It illustrates the paired helical nature of the transmembrane segments and the elongated structures connecting them to the spike projecting domains. A 10 A diameter density in the fusion protein lines the cavity at the center of the spike. These clearly visible features combine with the variation in order between the layers to provide a framework for understanding the structural changes during the life cycle of an enveloped virus.

The structure of the particle formed by the SFVmSQL mutant of Semliki Forest virus (SFV) has been defined by cryo-electron microscopy and image reconstruction to a resolution of 21 A. The SQL mutation blocks the cleavage of p62, the precursor of the spike proteins E2 and E3, which normally occurs in the trans-Golgi. The uncleaved spike protein is insensitive to the low pH treatment that triggers membrane fusion during entry of the wild-type virus. The conformation of the spike in the SFVmSQL particle should correspond to that of the inactive precursor found in the early stages of the secretory pathway. Comparison of this "precursor" structure with that of the mature, wild-type, virus allows visualization of the changes that lead to activation, the first step in the pathway toward fusion. We find that the conformational change in the spike is dramatic but localized. The projecting domains of the spikes are completely separated in the precursor and close to generate a cavity in the mature spike. E1, the fusion peptide-bearing protein, interacts only with the p62 in its own third of the trimer before cleavage and then collapses to form a trimer of heterotrimers (E1E2E3)3 surrounding the cavity, poised for the pH-induced conformational change that leads to fusion. The capsid, transmembrane regions and the spike skirts (thin layers of protein that link spikes above the membrane) remain unchanged by cleavage. Similarly, the interactions of the spikes with the nucleocapsid through the transmembrane domains remain constant. Hence, the interactions that lead to virus assembly are unaffected by the SFVmSQL mutation., (Copyright 1998 Academic Press.)

Time-resolved cryoelectron microscopy reveals the first step in the conformational changes that enable membrane fusion in Semliki Forest virus. The neutral pH structure reveals a central cavity within the spike complex, plate-like extensions forming a layer above the membrane, and the paths of the paired transmembrane domains connecting the trimeric spikes and pentamer-hexamer clustered capsid subunits. Low pH treatment results in centrifugal movement of E2, the receptor-binding subunit, centripetal movement of E1 to narrow the central cavity initiating the formation of an E1 trimer, and the extension of the E1 fusion sequence toward the target membrane.