protocols for the purification of plant viruses

TECHNIQUES IN PLANT VIROLOGYCIP Training Manual5.0 VIRUS PURIFICATION

Section 5.2Protocols for thePurification of Plant Viruses

Purified virus preparations are needed to study the physical andbiochemical properties, to produce specific antisera, to prepare nucleicacid probes complementary to the viral sequence, and to determinetranslation products. Generally, purification consists of the followingstages:

P.V. • Sec 5.2 –99 • - INTERNATIONAL POTATO CENTER

Once the virus is purified, its concentration must be determined. For thispurpose the virus is diluted (usually 1/20) and read at absorbency OD =

Extraction

Clarification(low speed)

Precipitation(low speed)


Sedimentation(high speed)


Density gradients centrifugation(high speed)

Fractionating

Sedimentation(high speed)

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260. The reading is then adjusted to the dilution level used and divided bythe virus extinction coefficient. This coefficient is characteristic for eachvirus. It represents the absorbency of a virus concentration of 0.1%(1mg/ml) with an optical path length of radiation of 1cm and a wavelengthof 260 nm. The extinction coefficient is symbolized as follows:

For instance, an APLV (bottom component) preparation diluted at 1/20has an OD260 value of 0.37 and an extinction coefficient of 9.6. The virusconcentration is therefore:

0.37 x 20 = 7.4

7.4/9.6 = 0.77 mg/ml

A simple calculation will show the total mg of virus obtained in thepurification and the virus yield per kg in the host plant.

Small samples should be taken at each step of the purification for viewingin the electron microscope. This determines the concentration of the virusand whether or not it is free of host plant components. Below are themost common virus purification protocols.

0.1% E

1 cm, 260 nm


POTATO VIRUS M (PVM)

Host plant Datura metel or Lycopersicon pimpinelifoliumHarvest 18–20 days after inoculationWorking temperature 4°C on ice

ExtractionHomogenize plant material with 2 volumes (w/v) of 0.1 M phosphate

buffer pH 8.0 and 0.5% 2-mercaptoethanol

Filter through 3 layers of guazeAdd 10% (v/v) carbon tetrachloride and 8% (v/v) 1-butanol

Emulsify for one minute

Clarify7800 g for 10 minutes

Remove pellet

Remove supernatant

Process supernatant

Process pelletResuspend overnight in 0.01M

phosphate buffer pH 7.5 (1/10 of theoriginal volume)

Sediment72,500 g for 90 minutes


Remove pellet

Remove supernatant

Remove pellet

Remove supernatant

Process supernatant

Process pelletResuspend overnight in0.01M phosphate bufferpH 7.5 (1/50 orig. vol.)

Process supernatant

Process pelletResuspend overnight in0.01M phosphate buffer

pH 7.5 (1/100 of theoriginal volume)

SedimentOn sucrose cushions 20% (w/v). 1 ml

per tube, 160,400 g for 90 minutes


Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in a swinging bucket rotor

96,500 g for 120 minutes

FractionateCollect the virus band in the gradient

Process fractionsDilute 1:3 in 0.01M phosphate buffer pH 7.5



Remove pellet Process supernatant


Centrifuge in performed density gradientsof cesium chloride (30, 60, and 90%) 128,000 g for 5 hours

FractionateCollect the virus-containing band in the gradient

Dialyze Overnighttwice with 2 liters 0.01 M phosphate buffer pH 7.5

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Read absorbencyin a spectrophotometer


POTATO VIRUS X (PVX)

Host plant Nicotiana glutinosaHarvest 20 days after inoculation

ExtractionHomogenize plant material (50 to 60 g) with 2 volumes (w/v) in 0.1M

phosphate buffer pH 8.0 with 0.2% of 2-mercaptoethanol and 10%ethanol

Filterthrough 3 layers of gauze

Clarify7800 g for 20 minutes at 4°C

Remove pellet

Remove pellet

Process supernatantAdd 1% Triton X-100. Stir 1 hour at

4°C

Process supernatantAdd 0.2 M NaCl and 4% PEG (MW6000 to 8000). Stir for 1 hour at 4°C.

Incubate for 1 hour at roomtemperature


Precipitate7800 g for 30 minutes


Remove supernatant

Remove pellet

Remove supernatant

Remove pellet

Process pellet (at 4°°°°C)Resuspend in 8 ml 0.05 M phosphate

buffer pH 8.0 with 1% of Triton X-100and rinse the tubes immediately with 2

ml of the same buffer

Process supernatant

Process pelletResuspend in 0.5 ml 0.05 M

phosphate pH 7.2 using a rotatingstirrer at 200 rpm overnight

Process supernatant


CentrifugeOn sucrose cushion 30% (w/v)


Clarify2000 g for 5 minutes at 4°C

Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.01 M phosphate buffer pH 7.2

with 0.01 M EDTA in a swinging bucket rotor96,500 g for 75 minutes



Remove supernatant

Remove pellet

Process pelletResuspend in 0.4 ml 0.01 M

phosphate buffer pH 7.2Stir 4ºC overnight

Supernatant

Process fractionsDilute in the same volume of 0.1M phosphate buffer pH

7.2



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Read absorbency in a spectrophotometer


POTATO VIRUS A (PVA)

Host plant Nicotiana occidentalisHarvest 18–20 days after inoculation. Use leaves with central vein

removedWorking temperature 4°C on ice

ExtractionHomogenize plant material with 2 volumes (w/v) of 0.2 M phosphate

buffer pH 8.0 and 0.5% 2-mercaptoethanol, 1M of urea and 0.01MEDTA


CentrifugeOn sucrose cushion of 30% (w/v) in 0.05M citrate bufferpH 6.2 with 0.01M EDTA at 72,500 g for 180 minutes

Remove pellet

Remove pellet

Remove supernatant

Process supernatantAdd 1% Triton X-100. Stir at

4°C for 3 hours

Process supernatant

Process pelletResuspend in 0.05M citrate buffer pH6.2 with 0.01M EDTA and 1% Triton

X-100. Stir at 4°C overnight



CentrifugeOn sucrose cushion of 30% (w/v) in 0.01M citrate buffer pH 6.2

with 0.01M EDTA 160,400 g for 150 minutes

Remove pellet

Remove supernatant

Process supernatant

Process pelletResuspend in 0.05M citrate buffer pH 6.2 with

0.01M EDTA at 4°C overnight




Centrifuge in density gradientsof sucrose (10 to 40%, v/v) in 0.01M citrate buffer pH 6.2 with

0.01M EDTA in a swinging bucket rotor. 128,000 g for 40minutes.




Remove supernatantProcess pellet

Resuspend in 0.01M phosphate buffer pH7.2, stirring gently for 6 hours at 4°C.


Remove pellet Supernatant

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POTATO VIRUS S (PVS)

Host plant: Chenopodium quinoa (Andean strain) or Nicotiana debneyii (other strain).Harvest: 18 to 20 days after inoculation.

ExtractionHomogenize the plant material with 2 volumes (w/v) in 0.1M phosphate

buffer pH 8.0 with 0.15% of 2-mercaptoethanol and 10% of ethanol

FilterThrough 3 layers of gauze


Remove pelletProcess supernatant

Add 4% of PEG (MW 8000) and 1% of Triton X-100.Stir at 4°C for 60 minutes



Resuspend in 0.05M phosphate buffer pH 8.0 (1/10 ofthe original volume) with 1 mM of EDTA

Clarify10,000 g for 20 minutes




Remove supernatant Resuspend in 0.05M phosphate buffer pH 8.0 with1 mM of EDTA



Centrifuge in density gradientsof sucrose (10 to 40% w/v) in 0.05M phosphate buffer pH 8.0 with

1mM EDTA in swinging bucket rotor.100,000 g for 60 minutes




Resuspend in 0.05M phosphate buffer pH 8.0 with1 mM of EDTA at 4°C overnight

CentrifugeOn sucrose cushion 20% (w/v) 160,000 g for 3 hours



Centrifuge in density gradientsof sucrose (10 to 40% w/v) in 0.01M phosphate buffer, pH 8.0 with

0.01M EDTA in swinging bucket rotor.128,000 g for 40 minutes




Resuspend with 0.01M phosphate buffer pH 8.0(final volume: 1 or 2 ml)



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POTATO VIRUS Y (PVY)

Host plant: Nicotiana occidentalisHarvest: 15 to 20 days after inoculation. Use leaves with central vein removed.

ExtractionHomogenize the plant material with 2 volumes (w/v) of 0.2M

sodium and potassium buffer pH 8.0 with 0.2% of2-mercaptoethanol and 0.01M of EDTA

Remove supernatant


Process pelletResuspend in 0.02M Na and K phosphate buffer pH 7.2with 1% of Triton X-100 (1/5 of the original volume) at

4°C overnight



Add 1% of Triton X-100Stir at 4°C for 120 minutes



Add 4% of PEG (PM 6000 to 8000) and 0.2M of NaClStir at 4°C for 60 minutes and incubate at room

temperature for 60 minutes



FractionateCollect the band, which contain the virus in the gradient

Process fractionsAdd an equal volume of 0.01M Na and K phosphate buffer pH 7.2

with 0.01M of EDTA



Centrifugeon sucrose cushion 30% (w/v) in 0.05M Na and K phosphate buffer

pH 7.2 (15 ml of sample for 5 ml 30% sucrose)72,500 g for 150 minutes


Resuspend in 0.01M Na and K phosphate buffer pH 7.2with 0.01M EDTA (1/30 of the original volume) at 4°C

for 4 hours



Add 0.5 g of CsCl for each 1.5 ml of sample

Centrifugeon cesium chloride gradients (30, 60, and 90%, w/v) in 0.01M Naand K phosphate buffer pH 7.2 with 0.01M of EDTA (1.5 ml of

sample for each 3 ml gradient) in swinging bucket rotor.128,000 g for 3 hours




Resuspend in 0.01M Na and K phosphate buffer pH 7.2(1/50 of the original volume) overnight at 4°C



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ANDEAN POTATO MOTTLE VIRUS (APMV) ANDANDEAN POTATO LATENT VIRUS (APLV)

Host plant: Hybrid Nicotiana clevelandii x Nicotiana bigelovii.Harvest: 18 to 20 days after inoculation.

ExtractionHomogenize the plant material with two volumes (w/v) of 0.2Msodium and potassium phosphate buffer pH 8.0 with 0.2% of 2-

mercaptoethanol and 5mM of MgCl2


Emulsify with 20% chloroform (v/v)



Add 0.2M of NaCl and 6% (w/v) of PEG (MW 6000–8000), mix and incubate at 4°C for 120 minutes


Process pelletResuspend with 0.05M sodium and potassium phosphate

buffer pH 8.0 with 5mM of MgCl2 (40% of originalvolume). Stir for 120 minutes

Remove supernatant



Remove pellet


Process supernatant

Centrifugeon a 20% sucrose cushion (w/v) 1 ml per tube


Process pelletResuspend in 0.05M Na and K phosphate pH 8.0

with 5mM of MgCl2 overnight at 4°CRemove supernatant



Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.05M Na and K phosphate buffer

pH 8.0 with 5 mM of MgCl2 in swinging bucket rotor.100,000 g for 3 hours

FractionateCollect the band that contains the virus in the gradient

Process the fractionsDilute in 0.05M Na and K phosphate

pH 8.0 and 5 mM of MgCl2


Remove supernatant

Process pelletResuspend in 0.05M Na and K phosphate

pH 8.0 with 5 mM of MgCl2

Process pelletResuspend in 0.001 M Na and K phosphate

pH 8.0

Remove supernatant

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Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.05M Na and K phosphate pH 8.0

with 5mM of MgCl2in swinging bucket rotor.

100,000 g for 3 hours


Process the fractionsDilute 1:1 (v/v) in 0.05 M Na and K phosphate

pH 8.0 with 5 mM of MgCl2



TOBACCO RINGSPOT VIRUS (TRSV)

Host plant: Hybrid Nicotiana clevelandii x Nicotiana bigeloviiHarvest: 28 days after inoculation

ExtractionHomogenize the plant material with 1 volume (w/v) of 0.07M

phosphate buffer pH 7.0 with 0.01M of EDTA and 0.1% ofthyoglicolic acid


Filterthrough 3 layers of gauze.

Emulsify with 1 volume chloroform

Remove pellet

ProcessStir well and incubate for 1 hour

Collect the aqueous phase

Process supernatant


Process supernatantAdd 6% PEG (MW 8000) and 0.3 M

of NaClStir for 1 hour at 4°C

Remove pellet


Process pelletResuspend in 10% of original volume 0.07 M phosphate

pH 7.0 with 0.01M of EDTA for 3 hoursRemove supernatant


Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.07M phosphate pH 7.0 in

swinging bucket rotor96500 g for 45 minutes


Sediment160400 g for 60 minutes

Remove supernatant Resuspend the pelletin 0.07 M phosphate pH 7.0

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POTATO LEAFROLL VIRUS (PLRV)

Host plant: Physalis floridana or Solanum tuberosumHarvest: 35 days after inoculation. Storage the leaves at –70°C, one day before purification

ExtractionGrind plant material to a powder with liquid nitrogen. Add 2 volumes

(w/v) of 0.1 M trisodic citrate pH 6.2 with 10mM of EDTA,1% Na2 SO3, 1.5% celluclast, and 0.1% of thioglycolic acid and

stir overnight at room temperature.

HomogenizePlant material in a blender

Filterthrough 3 layers of gauze. Emulsify with 25% ofthe initial volume, of chloroform-1-butanol (1:1)

for 5 minutes

Remove pellet

Remove supernatant

Process supernatantAdd 0.4 M of NaCl and 8% of PEG(MW 6000–8000) and stir 2 hours at

room temperature

Process pelletResuspend in 0.01 M sodium

phosphate buffer pH 7.6.Overnight at 4°C.




Remove pellet

Remove supernatant

Remove supernatant

Process supernatant

Process pelletResuspend in 10mM sodium

phosphate buffer pH 7.6.Overnight at 4°C

Process pelletResuspend in 10 mM sodium

phosphate buffer pH 7.6.Overnight at 4°C


Centrifugeon sucrose cushion (20% w/v) in 10 mM sodium

phosphate pH 7.6.72530 g for 120 minutes

Centrifuge on sucrose cushion (20% w/v) in 10 mM sodium phosphate

buffer pH 7.672530 g for 120 minutes






Remove pellet

Remove pellet

Process supernatant

Process supernatant


Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 10 mM sodium phosphate buffer,

pH 7.6 in a swinging bucket rotor114500 g for 45 minutes

FractionCollect the band that contains virus in the gradient. Dilute

in 10mM sodium phosphate buffer pH 7.6



Remove supernatant


phosphate buffer pH 7.6Shake overnight at 4°C

Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 10 mM sodium phosphate buffer

pH 7.6 in a swinging bucket rotor114500 g for 45 minutes


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FractionateCollect the band that contains virus in the gradient. Dilute

in 10mM sodium phosphate buffer,pH 7.6


Remove supernatant


phosphate buffer pH 7.6Overnight at 4°C


POTATO VIRUS T (PVT)

Host plant: Chenopodium quinoaHarvest: 15 to 20 days after inoculation

ExtractionHomogenize plant material with 2 volumes (w/v) in 0.06 M phosphate

buffer, pH 7.0.

FilterThrough 3 layers of gauze.

Remove pellet Process supernatantFilter through glass wool.

Add 5% PEG (MW 8000). Stir for 1 hourat 4°C. Remove from shaker and let sit at

room temperature for 10 minutes.



Remove supernatant

Process pelletResuspend in 0.06 M

phosphate buffer, pH 7.0Overnight at 4°C






Resuspend in 0.06 Mphosphate buffer, pH 7.0

Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.06 M phosphate buffer, pH 7.0 in




Remove supernatantResuspend pellet

in 0.06 M phosphate bufferpH 7.0. Overnight at 4°C

Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.06 M phosphate buffer, pH 7.0 in





Remove supernatantResuspend pellet

in 0.06 M phosphatebuffer, pH 7.0. Overnight

at 4°C

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protocols for the purification of plant viruses

Documents

swinging bucket rotor

international potato center

hybrid nicotiana clevelandii

process pellet resuspend

potato virus

extraction homogenize

process supernatant

density gradients