You are on page 1of 10

TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual


5.0 VIRUS PURIFICATION

Section 5.1
Fundamentals of
Purification of Plant Viruses

Knowledge about a virus, before being purified, is very limited. Very pure
preparations of viruses are required in order to carry out chemical,
physical, and other biological studies. There are numerous purification
procedures that can be adapted to many of the viruses that infect plants.
However, there are several different purification systems that can be
selected for use according to the type of virus.

Group 1. A Sole Nucleoprotein Component

Virus particles of this group contain one molecule of central nucleic acid
(which contains the genetic code) surrounded by a protein capsid. Rod
shaped viruses such as tobacco mosaic and some spherical viruses such
as potato leafroll and sowbane mosaic belong to this group.
Group 2. Multicompound Nucleoproteins

Besides characteristic nucleoprotein particles, many viruses have other


particles that do not contain nucleic acid. Other viruses can possess
several different particles containing different quantities of nucleic acid.
Methods used for the purification of such viruses are very similar to those
used in Group 1, but in the final step, the different types of viral particles
are separated by taking advantage of the differences that exist in their
sedimentation coefficients or densities.

Group 3. Satellite Viruses

Some viruses are incapable of replicating themselves in a host unless the


host is simultaneously infected by another virus (e.g., the satellite virus of
tobacco necrosis virus). In this system, both viruses multiply together,
and are separated during or after purification.

Group 4. Enveloped Viruses

Besides the protein capsid that encloses the nucleic acid, these viruses
have an external membrane composed of lipids. These lipid membranes
are very delicate and are also easily damaged, which can cause the
inactivation of the particles. The procedures of virus purification in Group
1, which are aimed at destroying membranes, are not suitable for these
viruses. More gentle methods must be used.

Group 5. Viroids

Viroids, like the potato spindle tuber viroid (PSTVd), do not have a protein
capsid. They are free molecules of nucleic acid. The methods of
purification are based on methods used for the isolation of nucleic acids.
They cannot be isolated using any of the procedures used for the four
groups mentioned above.

Although there are many different methods of purification to isolate


viruses, there are some steps that they all have in common:

a. Viruses must multiply in an appropriate host, in which they reach


high concentrations.

b. Viruses must be extracted and placed in a liquid medium, with a


minimal loss of infectivity.

c. The extract must be clarified to remove most of the host material.

d. The isolated virus must be concentrated to a smaller volume.

Purification must always be carried out with biologically pure viruses.

This means that virus cultures must be free of other contaminating


viruses. Viruses mutate very rapidly and it is very difficult to avoid the
production of mutants in a culture. However, the mutation rate of viruses
can be kept to a minimum, eliminating the conditions in which they are
more frequent (such as high temperatures, irradiation, and exposure to

P.V. • Sec5.1 – 99 • Page 2 - INTERNATIONAL POTATO CENTER


chemical mutagenic components). The isolated virus must be regularly
analyzed by using indicator plants to determine the existence of mixtures.

The virus culture can be kept biologically pure by inoculating indicator


plants (in which local lesions are produced), isolating one lesion, and
inoculating another host plant with it. Transfer of various local lesions
reduces the probability that the virus is contaminated with another virus
or another virus strain. Once the culture is pure, all precautions must be
taken to prevent its contamination.

Selection of Host for Purification

Since virus concentration can change considerably in different host


plants, it is important to select a host plant from which great quantities of
virus can be obtained. Plants systemically infected with the virus are
always preferred to those in which the virus is localized.

The facility and speed of spreading in a host plant and the growth stage
in which they can be inoculated are very important factors to take into
account during selection.

The selected host plants selected should not contain great quantities of
tannins, gums, or phenolic compounds, because such compounds
interfere with virus purification. Generally, great quantities of these
compounds are found in ligneous plants, which makes them
inappropriate for purification.

The conditions under which inoculated plants grow determine the


concentration of virus in them. Maximum results are obtained in plants
that have vigorous growth. Environmental conditions such as
temperature and light affect virus concentration. Usually low
temperatures delay virus multiplication, while high temperatures induce
its mutation.

Usually, a temperature of 20–25°C is appropriate. Low light intensities


also help to induce virus multiplication. In regions where light intensity is
higher, reduction of light intensity to 20% of the external normal
illumination in the greenhouse allows the multiplication of the virus inside
the host. The nutrients of plants and day length may affect virus
multiplication; however, their effects are not as important as the effects of
light and temperature.

Certain plant viruses multiply in the leaves during a long period of time,
while others reach a concentration peak within a short period. With
certain viruses, concentration increases up to a certain level and then
starts to decrease. It is important to harvest the leaves when the
concentration level is close to the maximum. It is also important to
consider that virus concentration in different parts of the plant can vary
considerably.

Harvested leaves can be frozen and stored at – 20°C. However, many


viruses are inactivated by freezing. If the virus is not inactivated, this
freezing process denatures certain proteins of the plant, facilitating
purification.

Extraction Medium

P.V. • Sec5.1 – 99 • Page 3 - INTERNATIONAL POTATO CENTER


It is very important that the extraction medium be in contact with the
destroyed cells immediately. Some of the buffer solutions commonly
used are citrate, phosphate, borate, and Tris-HCl. For the extraction of
plant sap, the following materials are needed:

• Mortar and pestle (used for small-scale extractions).


• Blenders and juice extractors.
• Grinders.

Stable viruses (such as the tobacco mosaic virus) do not require a


complex medium for purification. However, many viruses have specific
requirements of pH and salt concentration. They may also need other
specific substances for stability.

1. pH

Concentration of hydrogen ions (pH) is an important factor, which


controls protein solubility. Charges in the proteins of the virus vary
according to the pH of the solution. The protein capsid of the virus
has a net charge of zero at its isoelectric point, and it precipitates.

Most viruses reach their isoelectric point in an acid medium. To


keep viruses in solution, the extraction medium must be alkaline.
However, the pH must not be too high. At high pH, bonds between
viral protein and nucleic acid are weakened: consequently, viral
particles swell and become more vulnerable to endonucleases
present in the sap of the plant.

2. Stabilizing Effect of Sap

In certain viruses, the crude extract may have a stabilizing effect,


and these viruses become less stable during purification. In such
cases the extraction medium has to be modified by adding
stabilizing substances during several stages of the purification
protocol.

Unstable viruses should be purified under cold conditions.

3. Metallic ions and molarity

To keep their infectivity, or to preserve the integrity of their


structure, some viruses need the presence of bivalent ions such as
++ ++
Ca or Mg . The molarity requirements vary depending on the
virus, some of them being stable at high molarities and others at
low molarities. The range of molarity can vary from 0.001 M to 0.5
M. In some cases, EDTA is added to minimize the aggregation
caused by bivalent metals. The EDTA is a chelating agent.

4. Plant components that inactivate the virus

P.V. • Sec5.1 – 99 • Page 4 - INTERNATIONAL POTATO CENTER


Generally, the harmful materials released during the
homogenization or extraction process can be reduced using large
amounts of a buffer solution.

Oxidation of phenolic compounds by copper-containing


polyphenoloxidase enzymes produce quinones, which can
aggregate or inactivate viruses. This can be prevented by adding
reducing agents such as sodium bisulfite, cysteine hydrochloride,
thioglycolic acid, ascorbic acid and mercaptoethanol, which
compete for the oxygen in the extract, or by adding copper
chelating agents such as DIECA-sodium salt (sodium
diethyldithiocarbamate), which inactivates the polyphenoloxidase.

The tannins existing in the plant extracts precipitate viruses. This


can be avoided using an alkaline pH, large amounts of buffer
solution, adding alkaloids (caffeine, nicotine sulfate), some
proteins (egg albumin, powder milk), and synthetic polymers
(polyvinylpyrrolidone). The alkaloids, the proteins, and the synthetic
polymers compete with the viruses to form aggregates with the
tannins.

5. Additives that eliminate proteins and ribosomes

Ribosomes have a sedimentation coefficient similar to that of the


viruses and must be eliminated using different additives. The
selection of the method depends on the stability of the viruses to
the additives used.

0.01M Na-EDTA pH 7.4 disassociates ribosomes, but can only be


used with viruses that do not need bivalent metallic ions to be
stable.

In some cases, bentonite can be used to eliminate 18s


ribosomes, proteins, and debris of chloroplasts.

7. Detergents

Triton x-100 can be used to help to liberate the viruses from those
insoluble cellular components such as membranes.

8. Enzymes

In those cases in which the virus is restricted to the phloem,


certain enzymes, such as pectinases and cellulases, can be used
to liberate the virus from the tissues.

Selective denaturation of host components

There are different methods to eliminate or to selectively denature the


host materials in the extract. The following are the most common:

1. Freezing

P.V. • Sec5.1 – 99 • Page 5 - INTERNATIONAL POTATO CENTER


The intact leaves or their extracts can be frozen. It is more
advantageous to freeze the extract instead of the tissue.

Repeated freezing and defrosting is more efficient than freezing


the extracts for long periods.

2. Heating

If the virus has a high thermal inactivation point, heating the


extracts at 40°C for an hour or at 50–60°C for 10 minutes will
denature the proteins from the plant. These denatured proteins
precipitate and can be easily separated.

3. Organic Solvents

Organic solvents that are partially soluble in water, such as


chloroform, butanol, and carbon tetrachloride, can be used to
denature the proteins from the plant. These organic compounds
denature lipoproteins as well. That is why they must not be used to
purify enveloped viruses (Group 4).

4. Selective Precipitation Using Salts and Alcohols

The viruses and proteins of the plants can be precipitated using


salts and alcohols. This can be used to selectively precipitate the
proteins of the plant and leaving the viruses in the solution. The
concentration of the salts required to precipitate the proteins
depends on the pH of the medium. The closer the pH is to their
isoelectric point, the more insoluble the host proteins and viruses.
Some plant proteins can be precipitated by reducing the pH to 5.0.
The proteins of the host plant can be selectively precipitated by
adding saturated ammonium sulfate to 40%.

Extract clarification

The initial extract usually contains big fragments of tissues, cells,


chloroplasts, nucleis, mitochondria, fragments of membranes, viruses,
proteins, and soluble salts.

The first things to eliminate are any particles larger than the virus, such
as chloroplasts, mitochondrias, etc. This is achieved by filtering the
extract.

An extract can be filtered through some layers of filter paper, many layers
of gauze, a fine nylon net, or glass wool to eliminate pieces of tissue and
unfragmented cells. The filtration can also be carried out through carbon,
diatomacrous earth, bentonite, cellulose powder, or Celite.

The use of glass wool and gauze is only effective to filter membranes and
big pieces of tissue. The filtrate still contains chloroplasts. A 10,0000-g
centrifugation for 10 minutes is usually enough to remove them. This
treatment produces sediment, which contains most of the big debris from
the plant. The viruses, certain kinds of ribosomes, particles smaller than
the viruses, proteins, and dissolved salts remain in solution.

P.V. • Sec5.1 – 99 • Page 6 - INTERNATIONAL POTATO CENTER


The clarified extract obtained can be processed by many different
methods to obtain the particles of the virus. Some of the methods used
are the following:

• Gel filtration (agar, agarose, sephadex)


• Crystallization
• Isoelectric precipitation
• Electrophoresis
• Ion exchange chromatography
• Two-phase systems
• Ultracentrifugation
• Centrifugation through density gradient
• Serological methods
• Dry gels
• Microfilters

The procedure used is chosen according to the stability of the virus and
the contaminants present in the clarified extract.

Crystallization, isoelectric precipitation, ultracentrifugation, and


centrifugation in density gradients are the most common methods used
for the potato viruses. However, this does not mean that the other
methods are not adequate.

1. Gel filtration (agar, agarose, sephadex)

This process is also known as molecular sieve chromatography


and is especially useful for those viruses that denature during high-
speed centrifugation.

The substance to be used as a molecular filter is packed in a


column. The smallest molecules enter the gel, while the larger
molecules are excluded and pass readily through the column. The
separation depends on the gel porosity and the size of the virus
particles as well as the cellular components of the plant (Debris).
The virus is obtained in a volume at least twice the original one,
and must be concentrated.

2. Crystallization

Many plant viruses and proteins can be precipitated or crystallized


due to the action of the salt concentration, especially if great
amounts of ammonium sulfate (25%) are added.

The resulting precipitate can be concentrated through filtration or


low-speed centrifugation. After that, the precipitate can be
dissolved in a small volume of buffer solution to obtain a
suspension of virus particles.

During crystallization, most of the crystallized or precipitated


proteins of the host are denatured and cannot be dissolved when
they are suspended again in a buffer solution. However, a certain
percentage of the host proteins do dissolve with the virus
particles.

P.V. • Sec5.1 – 99 • Page 7 - INTERNATIONAL POTATO CENTER


Generally, this process is not used in modern purification methods
because certain viruses are sensitive to high salt concentrations.

3. Isoelectric precipitation

It is also known as precipitation by acidification or by pH change.

Viruses are amphoteric molecules (which contain positive and


negative charges at the same time). There is a pH value known
as the isoelectric point, where the net charge of the molecule
becomes zero and will precipitate out of solution.

This procedure for concentrating viruses (lowering the pH to their


isoelectric point) is most useful for viruses that precipitate at a pH
value lower than 4.5, since most plant material precipitates at a
pH value of 4.8 or 5 and can thus be removed from the
preparation before the virus itself is precipitated.

4. Electrophoresis

This separation method is based on the migration of charged


particles or molecules through an electric field. The rate of
movement depends on, among other factors, the net charge of
the particle or molecule, the potential gradient formed by the
electric field, and the resistance of the medium to the movement
of the particle or molecule.

Electrophoresis in gels (semi-solid medium) of agarose or


agarose/acrylamide can be used for purifying viral preparations,
because viruses are charged particles that migrate through an
electric field (except at their isoelectric point). However, this
technique is not widely used for purifying plant viruses due to the
small quantities that must be used.

The electrophoresis in a density gradient column (a liquid


medium, usually sucrose) is more commonly used because a
much larger volume can be processed. This method is useful
when purifying unstable viruses or viruses that have a
sedimentation coefficient similar to that of vegetal components.

5. Chromatography of ionic exchange

Resins of ionic exchange are synthetic polymers, which are


produced as spheres with a certain number of ionizable groups.
One of these groups becomes fixed to the polymer and others
remain free to be replaced or exchanged. In resins of cationic
exchange, the fixed ion is negatively charged (anion) and the
positive ion (cation) can be exchanged. The opposite occurs in
resins of anionic exchange. According to the charge, molecules in
the virus preparation are retained or they pass freely. Although ion
exchange chromatography separates molecules with similar
electric charges, it is not commonly used to purify viruses
because it does not always separate the virus completely and
sometimes, the virus remains irreversibly joined to the resin. It is
normally used to remove some impurities from the suspension of
viruses.

P.V. • Sec5.1 – 99 • Page 8 - INTERNATIONAL POTATO CENTER


6. Two-phase systems

Also known as partition chromatography.

Virus purification using two liquid phases has been used up to a


limited extent. Examples of phase systems are: polyethylene
glycol-dextran and polyethylene glycol-dextran sulfate. The
process depends on salt concentration, and in some cases the
virus can be concentrated in the phase of the least volume.

7. Ultracentrifugation

Ultracentrifugation in a fixed angle rotor is the most commonly


used method to concentrate viruses. The viral particles sediment
against the sloping outer walls of the tube and slide to the bottom
to form a “pellet." Generally, this process takes 2 hours or less. It
is important to centrifuge the preparation during the least possible
time because sedimentation at high speed is a severe physical
process that may damage the virus particles. Immediately after
centrifugation, the supernatant (liquid) must be eliminated and the
sediment (pellet) resuspended in a small volume of buffer.

8. Centrifugation in density gradients

This technique is most commonly used for final virus purification.


Concentrated preparations of virus are carefully layered onto the
surface of tubes that contain pre-formed gradients and
centrifuged. During the centrifuge run, the virus and the host plant
components move along the gradient at different rates. After
centrifugation, the layer containing the virus may be observed
under diffuse light as a dense opalescent band and can be
collected from the tube by using a hypodermic syringe or using a
fractionator of density gradients.

9. Serological methods

There are two serological methods for concentrating purified


viruses and eliminating host plant contaminants.

The first is affinity chromatography. In this case, the virus


preparation is passed through a column with antiviral antibodies
adsorbed to the solid matrix. Viral particles are caught by the
antibodies and remain the column, while vegetal material is eluted
out. Viral particles are subsequently removed from the column by
dissociating the virus-antibody complex. This technique has been
successfully used in the preparation of highly pure viruses for the
production of antibodies.

The second method consists in using antiserum to remove host


proteins. The antiserum is prepared by injecting purified host plant
proteins into a rabbit. The antiserum is then purified and the
gammaglobulin fraction incubated with the viral preparation. The
antibody-host plant protein complex is removed by low speed
centrifugation and the viral preparation is separated from the
serum soluble proteins by centrifugation with density gradients.

P.V. • Sec5.1 – 99 • Page 9 - INTERNATIONAL POTATO CENTER


10. Adsorbent or dry gels

Calcium phosphate is one of the most commonly used adsorbents


for the purification of plant viruses. Freshly calcium phosphate
precipitates is obtained by mixing 1M CaCl2 and 0.2M Na2HPO4.
This precipitate must be washed several times in order to remove
residual salts and then it is mixed with the viral preparation. The
adsorbent-protein mixture is then separated by centrifugation or
filtration. Either host proteins or the virus can be adsorbed. The
virus can be eluted under appropriate conditions.

11. Microfilters

This technique has been widely used for the purification of animal
viruses. Systems of filters with pores of different sizes can be
arranged so that pre-filtration (to remove the biggest particles that
can clog filters with small pores) and virus concentration occur
simultaneously. This method is suitable for globular molecules and
for inflexible rod-shaped viruses such as the tobacco mosaic. It is
not recommended to use ribbon-shaped flexible molecules,
because they can be broken by the torsion force of filtration and
their fragments can clog the filters. Another disadvantage is that
viruses can stick on the filters of pre-filtration. One way of avoiding
virus loss consists in using filters with a pore diameter at least ten
times bigger than the size of the virus to be filtered.

P.V. • Sec5.1 – 99 • Page 10 - INTERNATIONAL POTATO CENTER