TECHNIQUES IN PLANT VIROLOGYCIP Training Manual5.0 VIRUS PURIFICATION

Section 5.1Fundamentals ofPurification of Plant Viruses

Knowledge about a virus, before being purified, is very limited. Very purepreparations of viruses are required in order to carry out chemical,physical, and other biological studies. There are numerous purificationprocedures that can be adapted to many of the viruses that infect plants.However, there are several different purification systems that can beselected 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. Rodshaped viruses such as tobacco mosaic and some spherical viruses suchas potato leafroll and sowbane mosaic belong to this group.

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Group 2. Multicompound Nucleoproteins

Besides characteristic nucleoprotein particles, many viruses have otherparticles that do not contain nucleic acid. Other viruses can possessseveral different particles containing different quantities of nucleic acid.Methods used for the purification of such viruses are very similar to thoseused in Group 1, but in the final step, the different types of viral particlesare separated by taking advantage of the differences that exist in theirsedimentation coefficients or densities.

Group 3. Satellite Viruses

Some viruses are incapable of replicating themselves in a host unless thehost is simultaneously infected by another virus (e.g., the satellite virus oftobacco 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 viruseshave an external membrane composed of lipids. These lipid membranesare very delicate and are also easily damaged, which can cause theinactivation of the particles. The procedures of virus purification in Group1, which are aimed at destroying membranes, are not suitable for theseviruses. More gentle methods must be used.

Group 5. Viroids

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

Although there are many different methods of purification to isolateviruses, there are some steps that they all have in common:

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

b. Viruses must be extracted and placed in a liquid medium, with aminimal 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 contaminatingviruses. Viruses mutate very rapidly and it is very difficult to avoid theproduction of mutants in a culture. However, the mutation rate of virusescan be kept to a minimum, eliminating the conditions in which they aremore frequent (such as high temperatures, irradiation, and exposure to

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

The virus culture can be kept biologically pure by inoculating indicatorplants (in which local lesions are produced), isolating one lesion, andinoculating another host plant with it. Transfer of various local lesionsreduces the probability that the virus is contaminated with another virusor another virus strain. Once the culture is pure, all precautions must betaken to prevent its contamination.

Selection of Host for Purification

Since virus concentration can change considerably in different hostplants, it is important to select a host plant from which great quantities ofvirus can be obtained. Plants systemically infected with the virus arealways preferred to those in which the virus is localized.

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

The selected host plants selected should not contain great quantities oftannins, gums, or phenolic compounds, because such compoundsinterfere with virus purification. Generally, great quantities of thesecompounds are found in ligneous plants, which makes theminappropriate for purification.

The conditions under which inoculated plants grow determine theconcentration of virus in them. Maximum results are obtained in plantsthat have vigorous growth. Environmental conditions such astemperature and light affect virus concentration. Usually lowtemperatures delay virus multiplication, while high temperatures induceits mutation.

Usually, a temperature of 20–25°C is appropriate. Low light intensitiesalso help to induce virus multiplication. In regions where light intensity ishigher, reduction of light intensity to 20% of the external normalillumination in the greenhouse allows the multiplication of the virus insidethe host. The nutrients of plants and day length may affect virusmultiplication; however, their effects are not as important as the effects oflight 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. Withcertain viruses, concentration increases up to a certain level and thenstarts to decrease. It is important to harvest the leaves when theconcentration level is close to the maximum. It is also important toconsider that virus concentration in different parts of the plant can varyconsiderably.

Harvested leaves can be frozen and stored at – 20°C. However, manyviruses are inactivated by freezing. If the virus is not inactivated, thisfreezing process denatures certain proteins of the plant, facilitatingpurification.

Extraction Medium

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It is very important that the extraction medium be in contact with thedestroyed cells immediately. Some of the buffer solutions commonlyused are citrate, phosphate, borate, and Tris-HCl. For the extraction ofplant 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 acomplex medium for purification. However, many viruses have specificrequirements of pH and salt concentration. They may also need otherspecific substances for stability.

1. pH

Concentration of hydrogen ions (pH) is an important factor, whichcontrols protein solubility. Charges in the proteins of the virus varyaccording to the pH of the solution. The protein capsid of the virushas a net charge of zero at its isoelectric point, and it precipitates.

Most viruses reach their isoelectric point in an acid medium. Tokeep viruses in solution, the extraction medium must be alkaline.However, the pH must not be too high. At high pH, bonds betweenviral protein and nucleic acid are weakened: consequently, viralparticles swell and become more vulnerable to endonucleasespresent 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 suchcases the extraction medium has to be modified by addingstabilizing substances during several stages of the purificationprotocol.

Unstable viruses should be purified under cold conditions.

3. Metallic ions and molarity

To keep their infectivity, or to preserve the integrity of theirstructure, some viruses need the presence of bivalent ions such asCa++ or Mg++. The molarity requirements vary depending on thevirus, some of them being stable at high molarities and others atlow molarities. The range of molarity can vary from 0.001 M to 0.5M. In some cases, EDTA is added to minimize the aggregationcaused by bivalent metals. The EDTA is a chelating agent.

4. Plant components that inactivate the virus

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Generally, the harmful materials released during thehomogenization or extraction process can be reduced using largeamounts of a buffer solution.

Oxidation of phenolic compounds by copper-containingpolyphenoloxidase enzymes produce quinones, which canaggregate or inactivate viruses. This can be prevented by addingreducing agents such as sodium bisulfite, cysteine hydrochloride,thioglycolic acid, ascorbic acid and mercaptoethanol, whichcompete for the oxygen in the extract, or by adding copperchelating agents such as DIECA-sodium salt (sodiumdiethyldithiocarbamate), which inactivates the polyphenoloxidase.

The tannins existing in the plant extracts precipitate viruses. Thiscan be avoided using an alkaline pH, large amounts of buffersolution, adding alkaloids (caffeine, nicotine sulfate), someproteins (egg albumin, powder milk), and synthetic polymers(polyvinylpyrrolidone). The alkaloids, the proteins, and the syntheticpolymers compete with the viruses to form aggregates with thetannins.

5. Additives that eliminate proteins and ribosomes

Ribosomes have a sedimentation coefficient similar to that of theviruses and must be eliminated using different additives. Theselection of the method depends on the stability of the viruses tothe additives used.

0.01M Na-EDTA pH 7.4 disassociates ribosomes, but can only beused with viruses that do not need bivalent metallic ions to bestable.

In some cases, bentonite can be used to eliminate 18sribosomes, proteins, and debris of chloroplasts.

7. Detergents

Triton x-100 can be used to help to liberate the viruses from thoseinsoluble 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 usedto liberate the virus from the tissues.

Selective denaturation of host components

There are different methods to eliminate or to selectively denature thehost materials in the extract. The following are the most common:

1. Freezing

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

Repeated freezing and defrosting is more efficient than freezingthe extracts for long periods.

2. Heating

If the virus has a high thermal inactivation point, heating theextracts at 40°C for an hour or at 50–60°C for 10 minutes willdenature the proteins from the plant. These denatured proteinsprecipitate and can be easily separated.

3. Organic Solvents

Organic solvents that are partially soluble in water, such aschloroform, butanol, and carbon tetrachloride, can be used todenature the proteins from the plant. These organic compoundsdenature lipoproteins as well. That is why they must not be used topurify enveloped viruses (Group 4).

4. Selective Precipitation Using Salts and Alcohols

The viruses and proteins of the plants can be precipitated usingsalts and alcohols. This can be used to selectively precipitate theproteins of the plant and leaving the viruses in the solution. Theconcentration of the salts required to precipitate the proteinsdepends on the pH of the medium. The closer the pH is to theirisoelectric 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 byadding 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, suchas chloroplasts, mitochondrias, etc. This is achieved by filtering theextract.

An extract can be filtered through some layers of filter paper, many layersof gauze, a fine nylon net, or glass wool to eliminate pieces of tissue andunfragmented 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 andbig pieces of tissue. The filtrate still contains chloroplasts. A 10,0000-gcentrifugation for 10 minutes is usually enough to remove them. Thistreatment produces sediment, which contains most of the big debris fromthe plant. The viruses, certain kinds of ribosomes, particles smaller thanthe viruses, proteins, and dissolved salts remain in solution.

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The clarified extract obtained can be processed by many differentmethods to obtain the particles of the virus. Some of the methods usedare 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 andthe contaminants present in the clarified extract.

Crystallization, isoelectric precipitation, ultracentrifugation, andcentrifugation in density gradients are the most common methods usedfor the potato viruses. However, this does not mean that the othermethods are not adequate.

1. Gel filtration (agar, agarose, sephadex)

This process is also known as molecular sieve chromatographyand is especially useful for those viruses that denature during high-speed centrifugation.

The substance to be used as a molecular filter is packed in acolumn. The smallest molecules enter the gel, while the largermolecules are excluded and pass readily through the column. Theseparation depends on the gel porosity and the size of the virusparticles 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 crystallizeddue to the action of the salt concentration, especially if greatamounts of ammonium sulfate (25%) are added.

The resulting precipitate can be concentrated through filtration orlow-speed centrifugation. After that, the precipitate can bedissolved in a small volume of buffer solution to obtain asuspension of virus particles.

During crystallization, most of the crystallized or precipitatedproteins of the host are denatured and cannot be dissolved whenthey are suspended again in a buffer solution. However, a certainpercentage of the host proteins do dissolve with the virusparticles.

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Generally, this process is not used in modern purification methodsbecause 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 andnegative charges at the same time). There is a pH value knownas the isoelectric point, where the net charge of the moleculebecomes zero and will precipitate out of solution.

This procedure for concentrating viruses (lowering the pH to theirisoelectric point) is most useful for viruses that precipitate at a pHvalue lower than 4.5, since most plant material precipitates at apH value of 4.8 or 5 and can thus be removed from thepreparation before the virus itself is precipitated.

4. Electrophoresis

This separation method is based on the migration of chargedparticles or molecules through an electric field. The rate ofmovement depends on, among other factors, the net charge ofthe particle or molecule, the potential gradient formed by theelectric field, and the resistance of the medium to the movementof the particle or molecule.

Electrophoresis in gels (semi-solid medium) of agarose oragarose/acrylamide can be used for purifying viral preparations,because viruses are charged particles that migrate through anelectric field (except at their isoelectric point). However, thistechnique is not widely used for purifying plant viruses due to thesmall quantities that must be used.

The electrophoresis in a density gradient column (a liquidmedium, usually sucrose) is more commonly used because amuch larger volume can be processed. This method is usefulwhen purifying unstable viruses or viruses that have asedimentation coefficient similar to that of vegetal components.

5. Chromatography of ionic exchange

Resins of ionic exchange are synthetic polymers, which areproduced as spheres with a certain number of ionizable groups.One of these groups becomes fixed to the polymer and othersremain free to be replaced or exchanged. In resins of cationicexchange, the fixed ion is negatively charged (anion) and thepositive ion (cation) can be exchanged. The opposite occurs inresins of anionic exchange. According to the charge, molecules inthe virus preparation are retained or they pass freely. Although ionexchange chromatography separates molecules with similarelectric charges, it is not commonly used to purify virusesbecause it does not always separate the virus completely andsometimes, the virus remains irreversibly joined to the resin. It isnormally used to remove some impurities from the suspension ofviruses.

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6. Two-phase systems

Also known as partition chromatography.

Virus purification using two liquid phases has been used up to alimited extent. Examples of phase systems are: polyethyleneglycol-dextran and polyethylene glycol-dextran sulfate. Theprocess depends on salt concentration, and in some cases thevirus can be concentrated in the phase of the least volume.

7. Ultracentrifugation

Ultracentrifugation in a fixed angle rotor is the most commonlyused method to concentrate viruses. The viral particles sedimentagainst the sloping outer walls of the tube and slide to the bottomto form a “pellet." Generally, this process takes 2 hours or less. Itis important to centrifuge the preparation during the least possibletime because sedimentation at high speed is a severe physicalprocess that may damage the virus particles. Immediately aftercentrifugation, the supernatant (liquid) must be eliminated and thesediment (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 thesurface of tubes that contain pre-formed gradients andcentrifuged. During the centrifuge run, the virus and the host plantcomponents move along the gradient at different rates. Aftercentrifugation, the layer containing the virus may be observedunder diffuse light as a dense opalescent band and can becollected from the tube by using a hypodermic syringe or using afractionator of density gradients.

9. Serological methods

There are two serological methods for concentrating purifiedviruses and eliminating host plant contaminants.

The first is affinity chromatography. In this case, the viruspreparation is passed through a column with antiviral antibodiesadsorbed to the solid matrix. Viral particles are caught by theantibodies and remain the column, while vegetal material is elutedout. Viral particles are subsequently removed from the column bydissociating the virus-antibody complex. This technique has beensuccessfully used in the preparation of highly pure viruses for theproduction of antibodies.

The second method consists in using antiserum to remove hostproteins. The antiserum is prepared by injecting purified host plantproteins into a rabbit. The antiserum is then purified and thegammaglobulin fraction incubated with the viral preparation. Theantibody-host plant protein complex is removed by low speedcentrifugation and the viral preparation is separated from theserum soluble proteins by centrifugation with density gradients.

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10. Adsorbent or dry gels

Calcium phosphate is one of the most commonly used adsorbentsfor the purification of plant viruses. Freshly calcium phosphateprecipitates is obtained by mixing 1M CaCl2 and 0.2M Na2HPO4.This precipitate must be washed several times in order to removeresidual salts and then it is mixed with the viral preparation. Theadsorbent-protein mixture is then separated by centrifugation orfiltration. Either host proteins or the virus can be adsorbed. Thevirus can be eluted under appropriate conditions.

11. Microfilters

This technique has been widely used for the purification of animalviruses. Systems of filters with pores of different sizes can bearranged so that pre-filtration (to remove the biggest particles thatcan clog filters with small pores) and virus concentration occursimultaneously. This method is suitable for globular molecules andfor inflexible rod-shaped viruses such as the tobacco mosaic. It isnot recommended to use ribbon-shaped flexible molecules,because they can be broken by the torsion force of filtration andtheir fragments can clog the filters. Another disadvantage is thatviruses can stick on the filters of pre-filtration. One way of avoidingvirus loss consists in using filters with a pore diameter at least tentimes bigger than the size of the virus to be filtered.