Cryo-electron tomography of vaccinia virus

Abstract

The combination of cryo-microscopy and electron tomographic reconstruction has allowed us to determine the structure of one of the more complex viruses, intracellular mature vaccinia virus, at a resolution of 4 -6 nm. The tomographic reconstruction allows us to dissect the different structural components of the viral particle, avoiding projection artifacts derived from previous microscopic observations. A surface-rendering representation revealed brickshaped viral particles with slightly rounded edges and dimensions of Ϸ360 ؋ 270 ؋ 250 nm. The outer layer was consistent with a lipid membrane (5-6 nm thick), below which usually two lateral bodies were found, built up by a heterogeneous material without apparent ordering or repetitive features. The internal core presented an inner cavity with electron dense coils of presumptive DNA-protein complexes, together with areas of very low density. The core was surrounded by two layers comprising an overall thickness of Ϸ18 -19 nm; the inner layer was consistent with a lipid membrane. The outer layer was discontinuous, formed by a periodic palisade built by the side interaction of T-shaped protein spikes that were anchored in the lower membrane and were arranged into small hexagonal crystallites. It was possible to detect a few pore-like structures that communicated the inner side of the core with the region outside the layer built by the T-shaped spike palisade.cryo-EM ͉ virus structure ͉ 3D reconstruction

The Product of the Vaccinia Virus L5R Gene Is a Fourth Membrane Protein Encoded by All Poxviruses That Is Required for Cell Entry and Cell-Cell Fusion

“…Recently, the presence of a single outer membrane bilayer was confirmed by freeze fracture (17) and was consistent with cryoelectron tomography (6), although the latter study suggested an additional membrane around the core. The fusion of the intracellular mature virion membrane with the plasma membrane was demonstrated by electron microscopy (2, 4) and supported by evidence for incorporation of viral membrane proteins in the plasma membrane (22) and lipid mixing studies (10).…”

Influenza virus pleiomorphy characterized by cryoelectron tomography

“…The virion structure is of interest not only in the context of virus assembly but also in light of the possibility that pleiomorphic variations may correlate with infectivity and/or pathogenicity. As a step toward addressing these questions, we have used cryoelectron tomography, a technique capable of rendering the 3D structures of individual macromolecular particles in their native states (19)(20)(21)(22)(23)(24)(25), to visualize influenza virions of the type A eggadapted X-31 strain (26).…”

Electron tomography of viruses

“…Studies on vaccinia virus in free and cell-associated states have been interpreted to provide evidence for a model in which cell attachment is required to induce decondensation of the viral DNA, and for a mechanism by which viral DNA is released into the cytoplasm by opening of the core on the side [4,34]. Electron tomographic studies on cells infected with flock house virus, a positive strand RNA virus, have been used to localize the 50 nm-sized compartments where viral RNA is synthesized and to delineate the architecture and stoichiometry of the viral replication complex [35].…”

“…However, the emergence of powerful technologies for carrying out electron tomography of viruses in isolation and in infected cells is rapidly beginning to provide novel glimpses of viral architecture in enveloped and complex viruses [4][5][6][7][8][9][10]. Here, we review some recent areas of progress and the challenges that lie ahead in this field.…”