Biology Reference
In-Depth Information
Chapter 1
Biophysical Methods to Monitor Structural Aspects
of the Adenovirus Infectious Cycle
Rosa Menéndez-Conejero, Ana J. Pérez-Berná, Gabriela N. Condezo,
Alvaro Ortega-Esteban, Marta del Alamo, Pedro J. de Pablo,
and Carmen San Martín
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 virol-
ogy, 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.
Key words Adenovirus structure, Adenovirus assembly, Adenovirus stability, Atomic force micros-
copy, Fluorescence spectroscopy, Electron microscopy, Immunolabeling, Ultrathin sections
1
Introduction
The quality of adenoviral preparations is usually judged from their
infectious titer (plaque assay or CPE detection), genome analysis
(restriction pattern or PCR), or transduction of visible markers
(e.g., GFP). Structural and biophysical techniques can provide
additional information regarding virion structure, assembly, and
stability, and can help determine the ultimate causes of compro-
mised infectivity in new constructs or preparations. Here we gather
together a collection of biophysical and imaging procedures that
represent alternative, complementary methods to evaluate the
structural integrity of an adenovirus preparation, or to elucidate
whether the viral cycle is impaired at a particular stage in the cell.
We begin with the determination of viral stability using fluores-
cence spectroscopy . Determination of the structural stability of a viral
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