Lecture 4: Physical characterization of Viruses
BSCI437
Different techniques used to study viruses:
 Agarose gel electrophoresis
 Polyacrylamide gel electrophoresis (PAGE)
-formamide or urea added to denature nucleic acids
-sodium dodecyl sulfate added to denature proteins (SDS-PAGE)
 ELISA
 Western Blot
 Northern Blot
 Southern Blot
 Column Chromatography
-molecular sieve
-ion-exchange
-affinity
 Centrifugation
 Ultracentrigfugation
-density gradient based (buoyant density)
-rate zonal (isokenetic)
Isolation, Detection, and Measurement of Viruses.
 Isolation. Given that viruses are not free living, they must be first isolated from a source. Classically,
sources may be the whole organism or a part thereof, excreted or secreted material, blood, or tissue.
Given the current state of the art, viruses can also be isolated by forensic methods.
 Samples are then typically processed. For detection of whole virus, this classically involves making a
suspension in a cold, physiological buffer, and centrifuging out the large debris and microorganisms.
For detection of virus by PCR, this would include preparative extraction of nucleic acids.
 Detection can be based on numerous methodologies.
 Clinical: the manifestation of some abnormality in host organisms or host cells.
 Epidemiological: Clinical but on the scale of populations.
 Diagnostic: Involves a test to physically detect the presence of virus. Methods include testing for the A)
presence of viral proteins (immunological based tests), viral nucleic acids (PCR-based), or for B) the
presence of active virus. These include assays for the formation of plaques, pocks, or foci.
 Measurement: Physical and functional methods to enumerate viruses.
 Physical Methods: Electron microscopy, optical density, Hemagglutination assay, various
immunoassays (e.g. ELISA, RIA), quantitative PCR.
 Functional Methods...the Infectious Unit: the number of viral particles it takes in order to establish an
infection
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Table of some Common techniques applied in Virology for studying viruses and viral components
Technique
Common (not necessarily
only) use
Resolving range and basis
Probe for locating
material (most common)
Agarose gel electrophoresis
Separation of DNA or RNA
0.1-60 kb (100-60,000 bases) depending on %
agarose. Based on size of nucleic acid.
Stain gel with ethidium bromide which
binds nucleic acid and fluoresces when UV
light is applied. Also use autoradiography if
nucleic acid is radioactive.
Polyacrylamide gel
electrophoresis (PAGE)
Separation of small DNA or RNA
molecules. Separation of proteins
About 5-2000 bases or base pairs for nucleic acid;
12-212 kDal (12,000-212,000 Daltons) for protein
depending on % polyacrylamide. Based on size,
shape, and charge (charge for proteins only)
Ethidium bromide for nucleic acids; stain
proteins with coomassie blue or silver to
visualize. Also autoradiography for
radioactive proteins or nucleic acids.
PAGE + Urea or
Formamide
Separation of single stranded DNA or
RNA
About 1-2000 bases depending on % polyacrylamide.
Based on size (number of bases).
See above for nucleic acid
PAGE + SDS (sodium
dodecyl sulfate)
Separation of proteins
About 12-212 kDal depending on % polyacrylamide.
Based on size (molecular weight) of protein.
See above for proteins on PAGE
Northern Blot
Location or detections of specific RNA
molecules among many. Transfer RNA
separated on agarose or polyacrylamide
gels to nitrocellulose or nylon filter
See above
Radioactive or flourescent labeled
oligonucleotide that binds to a specific
sequence of nucleic acid
Southern Blot
Location or detections of specific DNA
molecules among many. Transfer DNA
separated on agarose or polyacrylamide
gels to nitrocellulose or nylon filter
See above
Radioactive or flourescent labeled
oligonucleotide that binds to a specific
sequence of nucleic acid
Western Blot
Location or detection of specific proteins
among many. Transfer protein separated
on polyacrylamide gels to nitrocellulose or
nylon filter
See above
Protein antibody that recognizes a specific
protein. Antibody can either by
radiolabeled or linked to and enzyme.
ELISA (Enzyme-linked
immunosorbant assay)
Detection or quantitation of a specific
protein
less than1 x 10-9 grams (1 ng) can be detected
Protein antibody that recognizes a specific
protein. Antibody is generally linked to an
enzyme that performs an activity that can be
measured for quantitation.
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Column Chromatography
(gel filtration or molecular
sieve)
Separation of protein (sometimes nucleic
acids also)
From approximately 5-1000 kDal depending on the
size of the pores in the sepharose or sephadex beads
used in the column. Separation based on size (also
shape to some extent) of protein.
Total protein can be detected using specific
colorimetric kits or by absorbency of UV
light in a spectrophotometer. Specific
proteins can be detected using antibodies or
an assay that detects an activity that only
that protein has.
Column Chromatography
(ion-exchange)
Separation of proteins
Proteins of all sizes can be separated based on the
charge (amino acid make-up) of the amino acid
groups on the surface of the protein and the charge of
the ligand groups linked to the column resin.
As above
Column Chromatography
(affinity)
Separation of proteins
As above except basis is the affinity of proteins for
the ligand group linked to the column resin. Group is
generally a molecule that particular proteins bind to
or an antibody.
As above
Centrifugation in aqueous
buffer
Separation of molecules (ex. RNA, DNA,
proteins), macromolecular structures (ex.
Ribosomes, mitochondria) or particles (ex.
viruses).
Particles ranging from about 5S to several thousand
S can be separated based on size (shape to a lesser
extent).Separation is performed in aqueous buffer.
This allows rapid separation of particles or molecules
with large differences in S-value. Advantage is
speed of separation while disadvantage is resolution.
For viruses, a plaque assay can be used (see
lecture)
Centrifugation (rate zonal
or isokenetic)
As above
Separation is performed in a more viscous buffer,
generally sucrose or glycerol density gradient.
Separation is slower but resolution significantly
improved.
As above for viruses
Centrifugation (buoyant
density or isopycnic)
As above
Separation is performed in media that can form a
density gradient ranging from low (about 1.1 g/ml) to
high (about 1.9 g/ml (water is 1 g/ml)) density
(typically CsCl). Molecules or particles stop moving
and form a band in the centrifuge tube when they
reach a point in the gradient at which the media
density equals their density. Separation based solely
on density.
As above for viruses
Autoradiography
Visualization of radiolabeled nucleic acids
or protein separated by electrophoresis.
Radioactive molecules can be located and quantified
since the radioactive particles emitted by the
molecule will blacken the film at the location where
the molecule is present.
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Electron microscopy
Electron microscopy
Visualization for counting or structural
analysis of particles (viruses for example),
cell structures, or other macrostructures.
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Generally magnification is 25,000-250,000 times
Particle can be seen on the electron
micrograph image generated by the electron
microscope
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Centrifugation as a purification and characterization procedure:
Ultracentrifuge- A centrifuge capable of generating large centrifugal fields by rotating
samples at 20,000-100,000 rpm. Centrifugal forces of greater than 100,000 X gravity can
be generated.
Sedimentation coefficient
 Rate at which a macromolecule sediments under a defined gravitational force.
 This parameter is influenced by both the molecular weight and shape of a
macromolecule (larger and more spherical sed. faster).
 The basic unit is the Svedberg (S) which is 10-13 sec.
 This value can be used to estimate molecular weights in conjunction with other
values.
Buoyant density-Density at which a virus or other macromolecule neither sinks nor floats
when suspended in a density gradient (e.g., CsCl2 or sucrose).
The Svedberg equation
s
v
 r
2

m(1  v  m )  (  p   m )

f
f
Where:
S= Sedimentation coefficient
v = velocity
= angular velocity (in radians/sec. 1 revolution = 2 radians)
r – radius, i.e. distance from center of rotation
m = mass (grams)
v = partical specific volume of particle (in nm)
r = density of solvent (g/cm3)
f = frictional coefficient between particle and solvent. For a globular protein, f ≈ 1 (fp =
frictional coeffieient of the particle; fm = frict. coeff. of solvent).
volume of the particle
Types of sedimentation medium:
1. Aqueous Buffer (Water based)- Can be used to separate molecules with widely
different S values (ex. Nuclei from ribosomes)
2. Sucrose or glycerol gradients or cushions (isokenetic or rate-zonal)-A fixed
concentration or a linear gradient of these agents in buffer is used . The
compounds increase the density and viscosity of the medium therefore, decreasing
the rate at which macromolecule sediment through them and preventing the
sedimentation molecules with densities less than the medium. General approach
is to pour a "cushion" of material at the bottom of the centrifuge tube and
centrifuge the virion onto the cushion (cushion need not always be used). By
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controlling the time and speed of centrifugation a significant purification can be
obtained. Since most macromolecules have greater densities than these mediums
separation is based on S values. This can be used to separate molecules with
relatively close S values.
3. CsCl gradient centrifugation (isopycnic or buoyant density)- A linear gradient of
these compounds in buffer is prepared in the centrifuge tube. As the
concentration of the compound is increased the density of the medium increases
in the tube. Density is low at the top and high at the bottom. Macromolecule
centrifuged through will form a band at a position equal to their buoyant density.
Useful for separating molecules of different densities even when the densities are
very close. Drawback is that CsCl can permanently inactivate some viruses.
Other techniques for separation:
Virions can also be separated from contaminants by electrophoresis and column
chromatography. Note that these methods are normally not used to separate virions but
are used to separate nucleic acids or proteins. However, they can also be used to separate
and purify virions in some cases. Both methods separate macromolecule on the basis of
charge and/or size characteristics depending on the method employed. Although virions
have a variety of charged macromolecule only those charged groups exposed to the
surface contribute to electrophoretic mobility or ion-exchange characteristics.
Chromatography can be ion exchange in which charged groups are ligated to the
chromatographic medium. The charged virus can then be bound to the medium and
eluted by increasing the concentration of a competing ion (example is elution off of
phosphocellulose with KCl). Molecular sieve chromatography can also be used by using
special agarose instead of dextran (used to construct cellulose and sephadex matrixes)
based matrixes to prepare beads. This allows for very large pores to be formed which
virus particles can enter.
Detection of Viral nucleic acids
Indirect: Blotting techniques. Southern blot detects DNA, Northern blots detect RNA
Direct: PCR based assays. Microarrays examine effects of infection on gene expression.
Assessing the purity of virions: A variety of methods may be used to assess the
homogeneity of the preparation
May include among others:
 Spectrophotometric analysis (shown above)-UV absorption at 260 and 280 nm.
This ratio (260/280) is a characteristic of a pure virus and is dependent on the
amount of nucleic acid and protein in the virus. The number can be used to
estimate the amount in the preparation. Nucleic acid absorbs light about twice as
well at 260 vs. 280 and vice versa for protein.
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Absorbance
Nucleic acid
Protein
240
260
280
300
nanometer of light (nm)


Serological methods-antibodies to viral proteins are used to characterize, detect,
or quantify virions. Antibodies can be made in several ways. Whole virus
(possibly attenuated (modified so can't cause disease) can be injected into animals
(rabbit or mouse) and monoclonal (single type of antibody generally recognizes a
single epitope) or polyclonal (several different antibodies that may recognize
several epitopes) .
A second approach is to purify or clone individual viral proteins and inject these
directly. Methods available for using antibodies include ELISA (Enzyme-linked
immunosorbent assay), RIA (radioimmune assay), RIPA (radioimmune
precipitation assay), western blotting, direct precipitation of virus with antibody,
neutralization of viral infectivity, complement fixation by the virus-antibody
complex, and others.

Electron microscopy -Method allows the visualization of single virus particles. It
is based on the principle of electron scattering. A beam of electrons is focused on
the sample. Electrons within the specimen will scatter the electron beam. The
scattering effect is enhanced by the presence of heavy, electron rich metal ions
(i.e. gold, platinum) within the sample. This is why the sample is coated with a
solution containing a heavy metal. Resolution in the nm range (10-9 meters) is
possible. Negative staining (sodium phosphotungstate or uranyl acetate that will
stain background but not the virus particles) or shadowing techniques (place
specimen on support and direct a vaporized heavy metal across the sample at an
angle. This creates a region where relatively little metal deposits just behind the
viral particle (resulting in a shadow).

X-ray crystallography- involves the analysis of crystallized virus. Virus crystals
are symmetrical structures composed of many isometric viruses. The atoms of the
crystal will diffract X-rays in a structure dependent manner. This approach has
been used to analyze the structure of the viruses at the molecular level.
Resolution at the Angstrom level (10-10 meters, in the bond length range) is
possible.
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