Biophysical Methods to Monitor Structural Aspects of the Adenovirus Infectious Cycle

Abstract

In this chapter we compile a battery of biophysical and imaging methods suitable to investigate adenovirus structural stability, structure, and assembly. Some are standard methods with a long history of use in virology, such as embedding and sectioning of infected cells, negative staining, or immunoelectron microscopy, as well as extrinsic fluorescence. The newer cryo-electron microscopy technique, which combined with advanced image processing tools has recently yielded an atomic resolution picture of the complete virion, is also described. Finally, we detail the procedure for imaging and interacting with single adenovirus virions using the atomic force microscope in liquid conditions. We provide examples of the kind of data obtained with each technique.

Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center

“…Then, the grids were washed 3 times with PBS and milli-Q water, and stained with saturated uranyl acetate [86]. All incubations were carried out at room temperature.…”

“…At the desired time post infection, the medium was removed and the cells were fixed with 2% glutaraldehyde and 1% tannic acid in 0.4 M HEPES pH 7.2 for 1.5 h at room temperature. Embedding in Epon was carried out as previously described [ 86 ]. Ultrathin sections (~70 nm) were collected on Formvar-coated nickel grids, stained with saturated uranyl acetate and lead citrate as described previously [ 86 ] and examined in a JEOL JEM 1230 transmission electron microscope at 100kV.…”

“…Embedding in Epon was carried out as previously described [ 86 ]. Ultrathin sections (~70 nm) were collected on Formvar-coated nickel grids, stained with saturated uranyl acetate and lead citrate as described previously [ 86 ] and examined in a JEOL JEM 1230 transmission electron microscope at 100kV. Mock infected cells were processed in the same manner as a control.…”

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

“…At the desired time postinfection, the medium was removed, and the cells were fixed with 2% glutaraldehyde and 1% tannic acid in 0.4 M HEPES, pH 7.2, for 1.5 h at room temperature. Embedding in Epon resin was carried out as previously described (57). Ultrathin sections (ϳ70 nm) were collected on Formvar-coated nickel grids, stained with saturated uranyl acetate and lead citrate as described previously (57), and examined in a JEOL JEM 1230 transmission electron microscope at 100 kV.…”

“…Embedding in Epon resin was carried out as previously described (57). Ultrathin sections (ϳ70 nm) were collected on Formvar-coated nickel grids, stained with saturated uranyl acetate and lead citrate as described previously (57), and examined in a JEOL JEM 1230 transmission electron microscope at 100 kV. The electron density of viral particles in sections was analyzed using Xmipp (58).…”