ULTRASTRUCTURAL STUDIES
Ultrastructural studies can be considered under
three areas: physical methods, chemical methods, and electron microscopy.
Physical measurements of virus particles began in the 1930s with the earliest
determinations of their proportions by filtration through colloidal membranes
of various pore sizes. Experiments of this sort led to the first (rather
inaccurate) estimates of the size of virus particles. The accuracy of these estimates
was improved greatly by studies of the sedimentation properties of viruses in
ultracentrifuges in the 1960s (Figure 1.4). Differential centrifugation proved
to be of great use in obtaining purified and highly concentrated preparations
of many different viruses, free of contamination from host cell components,
that can be subjected to chemical analysis. The relative density of virus particles,
measured in solutions of sucrose or CsCl, is also a characteristic feature,
revealing information about the proportions of nucleic acid and protein in the
particles.
Figure 1.4 A
number of different sedimentation techniques can be used to study viruses. In
rate-zonal centrifugation (shown here), virus particles are applied to the top
of a preformed density gradient, i.e., a sucrose or salt solution of increasing
density from the top to the bottom of the tube (top of figure). After a period
of time in an ultracentrifuge, the gradient is separated into a number of
fractions, which are analysed for the presence of virus particles. In the
figure, the nucleic acid of the virus genome is detected by its absorption of
ultraviolet light (below). This method can be used both to purify virus
particles or nucleic acids or to determine their sedimentation characteristics.
In equilibrium or isopycnic centrifugation, the sample is present in a
homologous mixture containing a dense salt such as caesium chloride. A density
gradient forms in the tube during centrifugation, and the sample forms a band
at a position in the tube equivalent to its own density. This method can thus
be used to determine the density of virus particles and is commonly used to
purify plasmid DNA.
The physical properties of viruses can be
determined by spectroscopy, using either ultraviolet light to examine the
nucleic acid content of the particle or visible light to determine its
light-scattering properties. Electrophoresis of intact virus particles has
yielded some limited information, but electrophoretic analysis of individual
virion proteins by gel electrophoresis, and particularly also of nucleic acid
genomes (Chapter 3), has been far more valuable. However, by far the most
important method for the elucidation of virus structures has been the use of
x-ray diffraction by crystalline forms of purified virus.This technique permits
determination of the structure of virions at an atomic level.
The complete structures of many viruses,
representative of many of the major groups, have now been determined at a
resolution of a few angstroms (Å) (see Chapter 2 ).This advancement
has improved our understanding of the functions of the virus particle
considerably; however, a number of viruses have proven to be resistant to this
type of investigation, a fact that highlights some of the problems inherent in
this otherwise powerful technique. One problem is that the virus must first be
purified to a high degree; otherwise, specific information on the virus cannot
be gathered. This presupposes that adequate quantities of the virus can be
propagated in culture or obtained from infected tissues or patients and that a
method is available to purify virus particles without loss of structural
integrity. In a number of important cases, this requirement rules out further
study (e.g., hepatitis C virus). The purified virus must also be able to form
paracrystalline arrays large enough to cause significant diffraction of the
radiation source. For some viruses, this is relatively straightforward, and
crystals big enough to see with the naked eye and which diffract strongly are
easily formed. This is particularly true for a number of plant viruses, such as
tobacco mosaic virus (which was first crystallized by Wendell Stanley in 1935)
and turnip yellow mosaic virus (TYMV), the structures of which were among the
first to be determined during the 1950s. It is significant that these two
viruses represent the two fundamental types of virus particle: helical in the
case of TMV and icosahedral for TYMV (see Chapter 2). In many cases, however,
only microscopic crystals can be prepared. A partial answer to this problem is
to use ever more powerful radiation sources that allow good data to be
collected from small crystals. Powerful synchotron sources that generate
intense beams of radiation have been built during the last few decades and are
now used extensively for this purpose; however, there is a limit beyond which
this brute force approach fails to yield further benefit. A number of important
viruses steadfastly refuse to crystallize; this is a particularly common
problem with irregularly shaped viruses—for example, those which have an outer lipid envelope—and to date no complete high-resolution atomic
structure has yet been determined for many viruses of this type (e.g., HIV).
Modifications of the basic diffraction technique (such as electron scattering
by membrane-associated protein arrays and cryo-electron microscopy) may help to
provide more information in the future, but it is unlikely that these
variations will solve this problem completely. One further limitation is that
some of the largest virus particles, such as poxviruses, contain hundreds of
different proteins and are at present too complex to be analysed using these
techniques.
Nuclear magnetic resonance (NMR) is increasingly
being used to determine the atomic structure of all kinds of molecules,
including proteins and nucleic acids. The limitation of this method is that
only relatively small molecules can be analysed before the signals obtained
become so confusing that they are impossible to decipher with current
technology. At present, the upper size limit for this technique restricts its
use to molecules with a molecular weight of less than about 30,000 to 40,000,
considerably less than even the smallest virus particles. Nevertheless, this
method may well prove to be of value in the future, certainly for examining
isolated virus proteins if not for intact virions.
Chemical investigation can be used to determine
not only the overall composition of viruses and the nature of the nucleic acid
that comprises the virus genome but also the construction of the particle and
the way in which individual components relate to each other in the capsid. Many
classic studies of virus structure have been based on the gradual, stepwise
disruption of particles by slow alteration of pH or the gradual addition of
protein-denaturing agents such as urea, phenol, or detergents. Under these
conditions, valuable information can sometimes be obtained from relatively
simple experiments. For example, as urea is gradually added to preparations of
purified adenovirus particles, they break down in an ordered, stepwise fashion
which releases subvirus protein assemblies, revealing the composition of the
particles. In the case of TMV, similar studies of capsid organization have been
performed by renaturation of the capsid protein under various conditions
(Figure 1.5). In simple terms, the reagents used to denature virus capsids can
indicate the basis of the stable interactions between its components. Proteins
bound together by electrostatic interactions can be eluted by addition of ionic
salts or alteration of pH; those bound by nonionic, hydrophobic interactions
can be eluted by reagents such as urea; and proteins that interact with lipid
components can be eluted by nonionic detergents or organic solvents.
In addition to revealing fundamental structure,
progressive denaturation can also be used to observe alteration or loss of
antigenic sites on the surface of particles, and in this way a picture of the
physical state of the particle can be developed. Proteins exposed on the
surface of viruses can be labelled with various compounds (e.g., iodine) to
indicate which parts of the protein are exposed and which are protected inside
the particle or by lipid membranes. Cross-linking reagents such as psoralens or
newer synthetic reagents with side-arms of specific lengths are used to
determine the spatial relationship of proteins and nucleic acids in intact
viruses.
Figure 1.5 The
structure and stability of virus particles can be examined by progressive
denaturation or renaturation studies. At any particular ionic strength, the
purified capsid protein of tobacco mosaic virus (TMV) spontaneously assembles
into different structures, dependent on the pH of the solution. At a pH of
around 6.0, the particles formed have a helical structure very similar to
infectious virus particles. As the pH is increased to about 7.0, disk-like
structures are formed. At even higher pH values, individual capsid monomers
fail to assemble into more complex structures.
Since the 1930s, electron microscopes have
overcome the fundamental limitation of light microscopes: the inability to
resolve individual virus particles owing to physical constraints caused by the
wavelength of visible light illumination and the optics of the instruments. The
first electron micrograph of a virus (TMV) was published in 1939. Over
subsequent years, techniques were developed that allowed the direct examination
of viruses at magnifications of over 100,000 times.The two fundamental types of
electron microscope are the transmission electron microscope (TEM) and the
scanning electron microscope (SEM) (Figure 1.6). Although beautiful images with
the appearance of three dimensions are produced by the SEM, for practical
investigations of virus structure the higher magnifications achievable with the
TEM have proved to be of most value. Two fundamental types of information can
be obtained by electron microscopy of viruses: the absolute number of virus
particles present in any preparation (total count) and the appearance and
structure of the virions (see below). Electron microscopy can provide a rapid
method of virus detection and diagnosis but in itself may give misleading
information. Many cellular components (for example, ribosomes) can resemble ‘virus-like particles,’ particularly in crude preparations. This difficulty can be overcome
by using antisera specific for particular virus antigens conjugated to
electron-dense markers such as the iron-containing protein ferritin or
colloidal gold suspensions. This highly specific technique, known as
immunoelectron microscopy, is gaining ground as a rapid method for diagnosis.
Figure 1.6
Working principles of transmission and scanning electron microscopes.
Developments in electron microscopy have allowed
investigation of the structure of fragile viruses that cannot be determined by
x-ray crystallography. These include cryo-electron microscopy, in which the
virus particles are maintained at very low temperatures on cooled specimen
stages; examination of particles embedded in vitreous ice, which does not
disrupt the particles by the formation of ice crystals; low-irradiation
electron microscopy, which reduces the destructive bombardment of the specimen
with electrons; and sophisticated image-analysis and image-reconstruction
techniques that permit accurate, three-dimensional images to be formed from
multiple images that individually would appear as very poor quality.
Conventional electron microscopy can resolve structures down to 50 to 70 Å
in size (a typical atomic diameter is 2–3 Å; a protein a-helix, 10 Å;
a DNA double helix, 20 Å). Using these newer techniques it is
possible to resolve structures of 25 to 30 Å.
In the late 1950s, Sydney Brenner and Robert
Horne (among others) developed sophisticated techniques that enabled them to
use electron microscopy to reveal many of the fine details of the structure of
virus particles. One of the most valuable techniques proved to be the use of
electron-dense dyes such as phosphotungstic acid or uranyl acetate to examine
virus particles by negative staining. The small metal ions in such dyes are
able to penetrate the minute crevices between the protein subunits in a virus
capsid to reveal the fine structure of the particle. Using such data, Francis
Crick and James Watson (1956) were the first to suggest that virus capsids are
composed of numerous identical protein subunits arranged either in helical or
cubic (icosahedral) symmetry. In 1962, Donald Caspar and Aaron Klug extended
these observations and elucidated the fundamental principles of symmetry, which
allow repeated protomers to form virus capsids, based on the principle of
quasi-equivalence (see Chapter 2). This combined theoretical and practical
approach has resulted in our current understanding of the structure of virus particles.
