The Surface Conformation of Sindbis Virus Glycoproteins E1 and E2 at Neutral and Low pH, as Determined by Mass Spectrometry-Based Mapping

Sindbis virus contains two membrane glycoproteins, E1 and E2, which are organized into 80 trimers of heterodimers (spikes). These trimers form a precise T54 icosahedral protein lattice on the surface of the virus. Very little is known about the organization of the E1 and E2 glycoproteins within the spike trimer. To gain a better understanding of how the proteins E1 and E2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification in combination with mass spectrometry. We have determined that at neutral pH the E1 protein regions that are accessible to proteases include domains 1‐21 (region encompassing amino acids 1 to 21), 161‐176, and 212‐220, while the E2 regions that are accessible include domains 31‐84, 134‐148, 158‐186, 231‐260, 299‐314, and 324‐337. When Sindbis virus is exposed to low pH, E2 amino acid domains 99‐102 and 262‐309 became exposed while other domains became inaccessible. Many new E1 regions became accessible after exposure to low pH, including region 86‐91, which is in the putative fusion domain of E1 of Semliki Forest virus (SFV) (M. C. Kielian et al., J. Cell Biol. 134:863‐872, 1996). E1 273‐287 and region 145‐158 were also exposed at low pH. These data support a model for the structure of the alphavirus spike in which the E1 glycoproteins are centrally located as trimers which are surrounded and protected by the E2 glycoprotein. These data improve our understanding of the structure of the virus membrane and have implications for understanding the protein conformational changes which accompany the process of virus-cell membrane fusion. Sindbis virus, the prototype of the Alphavirus subgroup of the Togaviridae, has a well-defined T54 icosahedral structure composed of three structural proteins E1, E2, and C (3, 10, 32, 49). The capsid protein combines with the virus’s positive-sense 42S RNA genome in the infected cell cytoplasm and is enveloped in a host-derived membrane bilayer which contains the two virus structural glycoproteins E1 and E2 (7, 8, 10, 47). The E1 and E2 glycoproteins form trimers of heterodimers in the endoplasmic reticulum of the infected cell. The trimers are exported to the plasma membrane, where interaction with nucleocapsids followed by lateral associations between 80 of the trimers produces the icosahedral lattice of spike complexes on the surface of the virus (3, 32, 36, 47). These protein-protein associations are responsible for the precise icosahedral structure of the virus membrane and are stable even in the presence of a nonionic detergent (36). The virus nucleocapsid is situated within the virus membrane and is composed of 240 copies of the capsid protein organized into a T54 icosahedron matching, precisely, the geometry of the glycoproteins in the virus membrane. The inner icosahedral protein shell is connected to the outer membrane protein shell through specific interactions between the capsid protein and the endodomain of glycoprotein E2 (22, 23). It has been difficult to study the configuration of the structural glycoproteins in the Sindbis virus membrane bilayer. No crystal structure exists for the membrane glycoproteins, and although the technology has greatly improved, it is unlikely that electron cryomicroscopy will produce images at a resolution sufficient to ascertain protein configurations at atomic