Lecture 4: Physical
characterization of Viruses
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
PCR
Column Chromatography
-molecular sieve
-ion-exchange
-affinity
Centrifugation
Ultracentrifugation
-density gradient based (buoyant density)
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-rate zonal (isokenetic)
Isolation and Detection, and
Measurement of Viruses
Isolation. Must first be isolated from a source, e.g.
•whole organism or a part thereof
•excreted or secreted material,
•blood,
•tissue.
Samples are then typically processed.
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.
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Measurement of Viruses
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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Common techniques applied in Virology for
studying viruses and viral components
Technique
• Separation of proteins and nucleic acids by
size
– Agarose gel electrophoresis
– Polyacrylamide gel electrophoresis (PAGE)
• PAGE + Urea or Formamide
• PAGE + SDS (sodium dodecyl sulfate)
• Detection of nucleic acids by autoradiography
– Northern Blot (RNA)
– Southern Blot (DNA)
– PCR
• Immunodetection
– Western Blot
– ELISA (Enzyme-linked immunosorbant assay)
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Common techniques applied in Virology for
studying viruses and viral components
Technique
• Column Chromatography (gel filtration or molecular
sieve)
– ion-exchange
– affinity
• Centrifugation
– in aqueous buffer
– rate zonal or isokenetic
– buoyant density or isopycnic
• Electron microscopy
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Centrifugation
• 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.
– Influenced by
• molecular weight
• shape of a macromolecule
– The basic unit is the Svedberg (S):10-13 sec.
– 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).
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v
The Svedberg equation
 (r p  rm )
m(1  v r m )
s 


2
 r
f
f
v
S= Sedimentation coefficient
v = velocity
w = angular velocity (in radians/sec. 1 revolution = 2p
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
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Sedimentation media
• Aqueous Buffer (Water based)-
– Used to separate molecules with widely different S
values (ex. Nuclei from ribosomes)
• Sucrose or glycerol gradients or cushions
(isokenetic or rate-zonal)–
–
–
–
Fixed concentration or linear gradients.
Increase the density and viscosity
Decreasing sedimentation rates
preventing the sedimentation molecules with densities
less than the medium.
– 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.
– Can be used to separate molecules with relatively close8 S
values.
Sedimentation media
• CsCl gradient centrifugation (isopycnic or
buoyant density)-
– 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 buoyant density.
– Useful for separating molecules of different
densities even when the densities are very close.
– Drawback: CsCl can permanently inactivate some
viruses.
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Other techniques for separation
Electrophoresis, column chromatography.
• Not normally used to separate virions but are used to
separate nucleic acids or proteins.
• Can be used to separate and purify virions in some cases.
• Both methods separate macromolecule on the basis of
charge and/or size characteristics.
• 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.
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• Molecular sieve chromatography allows for very large pores
to be formed which virus particles can enter
Electrophoresis
Size exclusion chromatography
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Indirect detection of viral nucleic acids
• Blots: after gel
separation, transfer
to solid phase and
probe with labeled
nucleic acids
– Southern Blot:
detects DNA
– Northern Blot:
detects RNA
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Southern blot (Fig 2.13)
Direct Detection of viral
nucleic acids
• PCR based assays: Directly amplifies
nucleic acids from sample
• Microarray technology: examines the
effects of viral infection on gene
expression
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Saiki, et al. 1988. Science 239: 487-491.
Revolutionized the molecular biosciences as we know them.
PCR: Exponential amplification
n
2
- 2(n-1) -2
duplex DNA molecules of the expected length
after n cycles of amplification (n = 1, 2, ...)
PCR- the method
Assessing the purity of virions
Spectrophotometric analysis
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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Assessing the purity of
virions
Serological methods
• Antibodies to viral proteins are used to:
– Characterize
– Detect
– Quantify virions.
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Antibodies can be made in
several ways.
• Whole virus or recombinant proteins
injected into animals
• Polyclonal, monoclonal
• Recombinant single chain antibodies
made in E. coli.
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Use of Antibodies:
direct and indirect
detection of viral
antigen.
(Fig. 2.8)
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Methods for using antibodies
•
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•
•
•
•
•
•
ELISA (Enzyme-linked immunosorbent assay),
RIA (radioimmune assay)
RIPA (radioimmune precipitation assay)
Colorometric bead-based diagnostic assays
Western blotting
Direct precipitation of virus with antibody
Neutralization of viral infectivity
Complement fixation by the virus-antibody
complex
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To detect viral proteins in a sample
ELISA:
Enzyme
Linked
Immuno
Sorbant
Assay
Fig. 2.11
To detect antibodies to a viral protein
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Immunoprecipitation (Fig. 2.9)
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Western blot (Fig. 2.10)
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Electron microscopy
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•
•
•
•
•
•
•
•
Allows the visualization of single virus particles.
Resolution in the nm range (10-9 meters) is possible.
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 within the sample.
SEM (Scanning EM) versus TEM (transmission EM)
Negative staining: stains background but not the virus
particles)
Shadowing techniques. Creates a region where
relatively little metal deposits just behind the viral
particle (resulting in a shadow).
Cryo-EM: freeze particles in vitreous ice, do SEM,
collect images, and reconstruct in 3-D
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T4 phage Negative staining
TEM of HIV
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Cryo-EM: freeze particles in vitreous
ice, do SEM, collect images, and
reconstruct in 3-D
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X-ray crystallography• Analysis of crystallized virus.
• Virus crystals are symmetrical structures
composed of many isometric viruses.
• Atoms of the crystal diffract X-rays in a
structure dependent manner.
• Resolution at the Å level (10-10 meters, in the
bond length range) is possible.
• This approach has been used to analyze the
structure of the viruses at the molecular
level.
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X-ray crystallography: atomic
resolution
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