Three-dimensional structure of a membrane-containing virus.

Abstract

The structure of Sindbis virus was determined by electron cryomicroscopy. The virion contains two icosahedral shells of viral-encoded proteins separated by a membrane bllayer of cellular origin. The three-dimensional structure of the Ice-embedded intact Sindbis virus, reconstructed from electron images, unambiguously shows that proteins in both shelas are arranged with the same icosahedral lattice of trianglation number T = 4. These studies also provide structural evidence ofcontact between the glycoprotein and the nucleocapsid protein across the membrane bilayer. The structural organization of Sindbis virus has profound implications for the morphogenesis of the alphaviruses. The observed interactions conflrm stoichiometric and speciflc protein associations that may be crucial for virlon stability and predict a mechanism for assembly.Membrane-containing viruses are assembled through the interaction of virus-encoded proteins with a host-cell membrane. For most enveloped viruses, this process involves two pathways. In one of these pathways, virus-encoded membrane proteins are cotranslationally integrated into membranes of the cell endoplasmic reticulum (1). These proteins are subsequently processed and delivered to a particular cell membrane (usually the plasma membrane) by a sequence of transport and processing events used by the cell in the maturation of its own membrane proteins. Virus membrane proteins, therefore, have proven to be important models for studying the processes involved in the maturation and in the targeting of cellular membrane proteins.While envelope proteins are transported to the plasma membrane, other viral proteins are translated in the cell cytoplasm and subsequently attach to the modified cellular membranes through the interaction of the cytoplasmic protein with cytoplasmic domains of the viral membrane glycoproteins (1). This association defines a critical step in the morphogenesis of the virus particle as it initiates and drives the process of envelopment of the core structure in the modified cellular membrane. The specific protein-protein interactions that occur during the fmal stages of assembly result in the production of a mature virus particle that maintains its structural integrity until its functional components interact with a potential host cell, and virus disassembly occurs.Sindbis virus, the prototype of the alphaviruses, achieves its mature structure in a distinctive fashion. Unlike many other enveloped viruses, the two-membrane glycoproteins of Sindbis virus (El and E2) are organized on the surface of the virus membrane as a precise triangulation number (T) = 4 icosahedron (2-5). The structure of this icosahedral lattice depends upon intramolecular disulfide bridges residing in the El glycoprotein (5, 6), and the integrity of the protein lattice has been demonstrated to determine the structure and stability of the membrane bilayer (6). Thus, whereas most enveloped viruses are described as including a membrane bilayer that contains virus-specified surface proteins an...

Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads

“…1d) as prototypes for particles bearing envelope proteins lying flat on the surface of the virus (SFV E1 and E2) and protruding from the bilayer (AIV HA and NA), respectively (Fujiyoshi et al, 1994;Harris et al, 2006). The SFVpp images did not reveal any additional protein density surrounding the outer leaflet of the lipid bilayer, although the envelope glycoproteins are reported to extend approximately 4-5 nm from the membrane (Mancini et al, 2000;Paredes et al, 1993), and despite immunogold labelling approaches revealing the presence of the E1 glycoprotein of SFV at the surface of SFVpps (data not shown). One plausible explanation is that the overall number of E1-E2 SFV complexes per particle was low and was therefore insufficient to provide enough contrast for their direct observation.…”

Nucleocapsid and Glycoprotein Organization in an Enveloped Virus

“…The organization of the nucleocapsid core subunits has been reported as either T = 3 (Horzinek and Mussgay, 1969;Fuller, 1987) or T = 4 (Coombs and Brown, 1987a;Paredes et al, 1993aParedes et al, , 1993b. A T = 3 structure would have nonequivalent associations between the inner nucleocapsid proteins and the envelope glycoproteins, whereas a T = 4 structure would permit stoichometric and quasiequivalent interactions between the nucleocapsid proteins and the glycoproteins.…”

Placement of the Structural Proteins in Sindbis Virus

“…The nucleocapsid cores are assembled in the cytoplasm of infected cells and are then transported to the plasma membrane, where they interact with the cytoplasmic domains of the glycoprotein spikes. During the budding process of the virion from the cell, the glycoproteins and a part of the plasma membrane are used to form the viral envelope to produce virions in which both the internal core and external glycoproteins have matching Tϭ4 icosahedral quasisymmetry (7,8,15,33,57). The mature Sindbis virus is composed only of the E1, E2, and capsid proteins.…”