A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia virus.

Abstract

We introduce a novel approach for combining immunogold labelling with cryoelectron microscopy of thin vitrified specimens. The method takes advantage of the observation that particles in suspension are concentrated at the air‐water interface and remain there during the subsequent immunogold labelling procedure. Subsequently, a thin aqueous film can be formed that is vitrified and observed by cryoelectron microscopy. In our view, a key early step in the assembly of vaccinia virus, the formation of the spherical immature virus, involves the formation of a specialized cisternal domain of the intermediate compartment between the endoplasmic reticulum and the Golgi. Using this novel cryoelectron microscopy approach, we show that in the intracellular mature virus (IMV) the core remains surrounded by a membrane cisterna that comes off the viral core upon treatment with dithiothreitol, exposing an antigen on the surface of the viral core. Complementary protease studies suggest that the IMV may be sealed not by membrane fusion but by a proteinaceous structure that interrupts the outer membrane. We also describe the structure and membrane topology of the second infectious form of vaccinia, the extracellular enveloped virus, and confirm that this form possesses an extra membrane overlying the IMV.

Cryo-electron tomography of vaccinia virus

“…The most prominent features derived from the tomographic reconstructions are a uniform size and shape of the particles, comprising a brick-shaped structure with the fixed dimensions of 360 ϫ 270 ϫ 250 nm, the presence of two membranes, lateral densities at both sides of the dumbbell core (whose membrane has a palisade composed of a layer of spikes), and electron-dense aggregations of DNA within the core. The apparent discrepancy found in the abundant literature reporting the shape of the cores from rounded to dumbbell-shaped (14,20,21) is explained by following the 3D volume of the reconstructed core ( Figs. 1 and 2).…”

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

“…The specimens were gently washed and then incubated with a 6-nm gold-conjugated secondary antibody for 1.5 hr at 37°C with 5% CO 2 . As with the studies of vaccinia virus assembly by Roos et al (1996), it was necessary to use gold-conjugated secondary antibodies as an electron-dense marker in order to resolve the locations of the primary antibodies and target proteins along the cell and viral surfaces.…”

Vaccinia Virus H3L Envelope Protein Is a Major Target of Neutralizing Antibodies in Humans and Elicits Protection against Lethal Challenge in Mice

“…An alternate maturation pathway is taken by a proportion of IMVs. They become wrapped in a double membrane from the transGolgi to form the intracellular enveloped virus, which then becomes translocated along microtubules to the cell surface, where the outermost intracellular enveloped virus membrane fuses with the plasma membrane (55). These virions may remain on the cell surface as cell-associated enveloped virus (CEV) or be released from the cell surface as EEV (7).…”