- Email: [email protected]
[97] Preparation and measurement of an antiviral protein found in tobacco cells after infection with tobacco mosaic virus
734
INTERFERON AND PLANT CELLS
[97]
Further Purification and Molecular Weight Estimation of IVR Further purification and estimation of the molecular weight were done by gel filtration through a Sephadex G-75 column. 7 A lyophilized IVR preparation obtained from 3 × 107 inoculated protoplasts was dissolved in I ml of 0.1 M phosphate buffer, pH 6.0 (OD280 = 2), placed on a precalibrated Sephadex G-75 column (33 × 2.3 cm), and eluted with the same buffer at a flow rate of 100 ml/hr. A control preparation obtained from a similar number of noninoculated protoplasts with the OD280 adjusted to 2 was passed through the same column in a similar way. Inhibitory potency of each third fraction, in comparison with the respective control fraction, was tested as before on inoculated Samsunn NN protoplasts. Peaks of activity were eluted at two positions, equivalent to molecular weights of 26,000 and 57,000. Characteristics of IVR IVR was stable in pH 2.5 and the antiviral activity was conserved after dialysis against 0.05 M phosphate buffer, pH 7.5, or against VIM. IVR was inactivated completely or its activity markedly reduced by treatment with trypsin or chymotrypsin, but not with ribonuclease? IVR was found to be thermolabile and was inactivated by heating at 60° for 10 min. These data suggest that IVR is of a proteineous nature. 7 j. R. Whitaker, Anal. Chem. 35, 1950 (1963).
[97] P r e p a r a t i o n a n d M e a s u r e m e n t o f a n A n t i v i r a l P r o t e i n F o u n d in T o b a c c o Cells a f t e r I n f e c t i o n w i t h Tobacco Mosaic Virus
By
ILAN SELA
AVF (antiviral factor) is a cumulative name given by Sela and Applebaum ~ to what turned out to be a group of closely related proteinaceous plant substances with antiviral activity. AVF, or its precursors, also present in noninfected tobacco leaves, is easily stimulated by various i 1. Sela and S. W. Applebaum, Virology 17, 543 (1962).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 119
Copyright (t) 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[97]
PLANT ANTIVIRAL PROTEIN
735
agents, 2 notably poly(I) • poly(C) 3 and especially TMV. 4 AVF stimulation is most pronounced following TMV infection of plants carrying the Ngene, which is responsible for localizing TMV infection in these plants 5,6 (for discussion of the N-gene see Sela2). A substance similar to AVF was later described in N-gene-carrying tobacco mesophyll protoplasts. 7,8 AVF, or some of it, is a phosphorylated glycoprotein. 9,~° Its activity in tissues, estimated at 0.1-10 molecules per cell, triggers some primary reactions whose products communicated to neighboring cells impose an "antiviral state."2 A major activity of AVF (and human leukocyte interferon) in tobacco cells is the stimulation of a synthetase activity which polymerizes ATP to oligoadenylates in the presence of dsRNA. 11 The plant oligonucleotides though antivirally active ~2 are not identical with 2,5-A,. 13,14AVF resembles interferon ~5which was indeed found to protect plant tissues from T M V , 16'17 as well as 2,5-A~ 18 and its derivatives. 19 Assaying Antiviral Activity in Plants General Comments on Quantitation
Measuring infectivity is a prerequisite for measuring antiviral activity. The lack of clonal cell lines in plants makes it impossible to develop a truly quantitative assay. Although a newly developed assay with protoplasts from standard tobacco cell suspensions, 2° is, so far, the best assay
2 I. Sela, Adv. Virus Res. 26, 201 (1981). 3 0 . Gat-Edelbaum, A. Altman, and 1. Sela, J. Gen. Virol. 64, 211 (1983). 4 TMV: tobacco mosaic virus. Y. Antignus. i. Sela, and I. Harpaz, J. Gen. Virol. 35, 107 (1977). ~' 1. Sela, A. Hauscbner, and R. Mozes, Virology 89, 1 (1978). 7 G. Loebenstein and A. Gera, Virology 114, 132 (1981), A. Gera, S. Spiegel, and G. Loebenstein, this volume [96]. Y. Antignus, 1. Sela, A. Hauschner, and !. Harpaz, Physiol. Plant Pathol. 6, 159 (1975). l0 R. Mozes, Y. Antignus, I. Sela, and I. Harpaz, J. Gen. Virol. 38, 241 (1978). ~t y . Devash, A. Hauschner, I. Sela, and K. ChakraburUy, Virology 111, 103 (1981). ~2 M. Reichman, Y. Devash, R. J. Suhadolnik, and I. Sela, Virology 128, 240 (1983). ~ Y. Devash, M. Reichman, 1. Sela, and R. J. Suhadolnik, Biochemist O, 24, 593 (1985). 14 y . Devash, I. Sela, and R. J. Suhadolnik, this volume [99]. is 1. Sela, Perspect. Virol. 11, 129 (1981). ~ P. Orchansky, M. Rubinstein, and I. Sela, Proc. Natl. Acad. Sci. U.S.A. 79, 2278 (1982). ~7 N. Rosenberg, M. Reichman, A. Gera, and 1. Sela, Virology 140, 173 (1985). t8 y . Devash, S. Biggs, and I. Sela, Science 216, 1415 (1982). ~9 y . Devash, A. Gera, M. Reichman, W. Pfleiderer, R. Charubala, I. Sela, and R. J. Suhadolnik, J. Biol. Chem. 259, 3482 (1984). -"~ I. Sela, M. Reichman, and A. Weissbach, Phytopathology 74, 385 (1984).
736
INTERFERON AND PLANT CELLS
[97]
for reliable quantitation of infection, it should still be considered as no more than an estimate. Measuring the antiviral activity of AVF and interferon is further complicated by the odd dose-response of tobacco tissues and cells. Interferon protects tobacco cells at a very low dosage, but its antiviral activity diminishes gradually with increase in interferon concentration. 17 With AVF at least two waves of antiviral activity are usually observed upon dilution.
Infectivity Assay: The Local Lesion Test Some viruses react with their host plants by producing local lesions (mostly necrotic) on the infected leaves. The number of local lesions is proportional to virus concentration, but, since leaves vary considerably in their response to viral infection, it is impossible to draw an accurate standard curve. In order to overcome this inherent shortcoming, at least partially, we usually combine the half-leaf test with one involving a dilution end-point. Purified TMV (prepared according to Devash et al. j~) is added to AVF or mock AVF 21 solutions to a final concentration of 5/~g TMV per ml 0.01 M sodium phosphate, pH 7.6, and carborundum (400600 mesh) is added to a concentration of 0.1 g/ml. The content of the tube is stirred with a sterile painter's brush which is then squeezed against the inner tube wall to remove excess liquid. Half-leaves of Datura stramenium L. are then inoculated by gently moving the brush from tip to petiol, creating 10-mm-wide inoculated strips. Control and AVF solutions are applied to opposite halves of the same leaves (Fig. I). A series of AVF dilutions to about 0.01 pg protein per ml are thus tested (the TMV concentration remains constant), each dilution being applied to 12 half-leaves. The plants are kept at 24 ° under constant illumination and local lesions are counted 3-4 days later. Activity at each dilution is expressed as a percentage of protection of the AVF-treated half-leaves compared to the control half-leaves.
The L e a f Disk Test In this test the viral content of leaf disks is determined either by the above described infectivity tests of disk homogenates, or, more reliably, by the enzyme-linked immunosorbent assay (ELISA). ~2 2i Mock AVF: A fraction prepared similarly to the particular AVF fraction but from noninfected plants. 22 M. F. Clark and A. H. Adams, J. Gen. Virol. 34, 475 (1977).
[97]
PLANT ANTIVIRAL PROTEIN
737
FIG. I. Half-leaf test. Leaves of D. stramonium were inoculated with 5 /zl/ml TMV containing 100 ng protein of mock DEAE-AVF preparation (left half) and 100 ng protein of DEAE-AVF (right half).
ELISA Reagents
PBS-Tween: 0.5 ml T w e e n 20 (Sigma), 0.2 g NaN3 in 1 liter PBS Coating buffer: 1.59 g Na2CO3, 2.93 g NaHCO3, 0.2 g NaN3 in 1 liter H20 P B S - T w e e n - P V P : 20 g PVP-40 (polyvinylpyrrolidone, Sigma) in 1 liter P B S - T w e e n P B S - T w e e n - P V P - B S A : 0.2% bovine serum albumin (BSA) in PSBTween-PVP Substrate buffer: 97 ml diethanolamine, 800 ml H20, 0.2 g NAN3. Titrate with HCI to pH 9.8 and bring to 1 liter with H20 Substrate solution: p-nitrophenyl phosphate. Keep solid at - 2 0 °. Prepare a fresh solution of 0.1 mg/ml in substrate buffer before each use Microplates: Dynatech 129B or Nunc Immunoplates
738
INTERFERON AND PLANT CELLS
[97]
ELISA Procedure The y-globulin fraction of chicken and rabbit sera immunized against TMV is prepared according to Clark and Adams z2 and stored at 1 mg/ml at - 2 0 °. To each well of a microtiter plate 200 /xl of chicken y-globulin, diluted to 1/zg/ml in coating buffer, is added and incubated for 3 hr at 30° or overnight at 4°. The wells are thoroughly washed (3 times) with PBSTween and 200/zl samples of TMV or disk homogenates (both prepared in PBS-Tween-PVP) are added to every well and incubated as above. Following washing, rabbit y-globulin (1 ~g/ml in PBS-Tween-PVP) is added to the wells and incubated as above. Commercially available goat antirabbit IgG (conjugated to alkaline phosphatase and diluted 1 : 1000 in PBSTween-PVP-BSA) is added next. Following incubation and washing as above, 250 ~1 of substrate solution is added and incubated at room temperature. Absorption at 410 nm is determined in a Microelissa Autoreader (Dynatech MR-580). Since a 96-well plate is read in less than a minute, the reaction is not stopped and readings are taken at 10 min intervals until 1 /zg/ml TMV yields an absorption of about 0.8. TMV standards for calibration curves are included in every plate, and every sample is applied in triplicate. Absorbance is linear with TMV concentration bewteen approximately 10 and 1000 ng/ml. TMV content is expressed as ng/disk. Since the TMV concentration must fall in the linear range, serial dilutions are made from each disk homogenate.
Antiviral Assay by ELISA Leaves of tobacco var. " S a m s u n " are dusted with carborundum and are inoculated with TMV (1/xg/ml in 0.01 M sodium phosphate, pH 7.6). After extensive washing with water, leaf disks (6 or 8 mm in diameter) are punched out with a paper-punch into a beaker containing the standard buffer. Twenty disks are collected at random from the common pool and placed in Petri dishes floating on 20 ml of the above buffer. Some plates contain disks from noninfected leaves, some contain untreated inoculated disks, and the rest contain the various materials to be tested for antiviral activity (added to the disks within 1 hr after inoculation). Plates are incubated at 24° under constant illumination. The disks are then frozen at - 2 0 °, homogenated with a Teflon-coated, motor-driven homogenizer, and their TMV content is determined by ELISA. TMV is first detected (in homogenate of 20 disks) 36 hr after inoculation, and increases rapidly between 48 and 96 hr after inoculation. It is better to follow the TMV growth curve in control and tested solutions, but since, in many cases, this is too elaborate a task, a single time-point between 60 and 80 hr is
[97]
PLANT ANTIVIRAL PROTEIN
739
D I L U T I O N OF CELL LYSATES
TREATMENT 1
1:2
1:4
1:8
1:16
1:32
FIG. 2. Dot-blot assay. TMV-inoculated protoplasts were treated with interferon (IFNaA; 0.1 units/ml) and with D E A E - A V F (60 ng protein/ml). Cell lysates were serially diluted (from left to right), spotted onto nitrocellulose, and hybridized as described in the text.
chosen. The homogenate of 20 disks, 72 hr after inoculation, should be diluted 1 : 10-1 : 100 for ELISA.
Antiviral Assay by Dot-Blot Test This test, developed in order to detect TMV-RNA in crude leaf and protoplast extracts, is about 100 times more sensitive than the ELISA. 2° This assay is based on a specific hybridization between radioactive cDNA (reverse-transcribed from TMV-RNA) and the RNA in the extracts and measures TMV-RNA accumulation. The detailed procedure is described elsewhere. 23 AVF and human interferon suppress TMV-RNA replication. The availability of highly purified interferon enables the expression of its activity in the protoplast system in terms of molecules per cell. By comparing cruder AVF preparations to interferon in the same experiment, interferon units could be assigned to AVF (Fig. 2). Purification AVF elutes as a broad peak of antiviral activity upon DEAE-cellulose chromatography and HPLC. After electrophoresis it appears as a broad z3 1. Sela, this volume [98].
740
INTERFERON AND PLANT CELLS
[97]
zone rather than a sharp band. The better resolving HPLC procedure exhibits, within the area of AVF activity, a number of protein peaks, some of which appear homogeneous on electrophoresis. Hence, it seems that AVF is a family of closely related molecules. The AVF preparations described herein cannot, therefore, be regarded as homogeneous. One HPLC fraction, which was studied a number of times was found to be homogeneous; however, it is premature to draw any definitive conclusions in this respect.
Reagents Standard buffer: 0.01 M sodium phosphate, pH 7.6 HCP (hydrated calcium phosphate24): a volume of 0.1 M Na2HPO4 is added to the same volume of 0. I M CaCI2. A precipitate is formed and washed 10 times with HzO by decantation. HCP is centrifuged prior to its addition to plant material to remove excess liquid Con A buffer: 0.1 M sodium acetate, pH 6.0; 1 mM MnCI2; 1 mM MgCI2; 1 mM CaCI2 HPLC buffer: 1 M pyridine, 2 M formic acid, pH 4.0
Protein Determinations A 50 /zl sample of an AVF preparation is mixed with 50 /xl of a Coomassie blue reagent 25,26 in an ELISA microtiter plate and the developed color is read at 570 nm in the automated ELISA reader. A calibration curve of known BSA concentrations is read in every plate. Green plant homogenate is precipitated with 7% perchloric acid and then redissolved in 0.1 N NaOH for protein determination. Dilutions (at least 1 : 10 in H20) of this basic solution can be added to the reagent and read quantitatively.
Plant Material Nicotiana glutinosa plants (carrying the N-gene) are grown in an insect-proof green house at 24 -+ 2° under constant illumination. At the stage of 4-6 fully expanded leaves and following dusting with carborundum, the leaves are rubbed gently with TMV (5 ~g/ml in the standard buffer). Leaves are harvested 2 days later when distinct local lesions appear but are not yet fully expanded.
24 R. W. Fulton, Virology 9, 522 (1959). 25 j. j. S e d m a k and S. E. Grossberg, Anal. Biochem. 79, 544 (1977). 26 Commercial kit with protocol from Bio-Rad Laboratories.
[97]
PLANT ANTIVIRAL PROTEIN
741
Procedure Step 1: Preparation of Crude AVF. The leaves are homogenized in a blender in the standard buffer at a ratio of 1:1 (w/v). The homogenate is squeezed through 3 layers of cheesecloth and centrifuged at 5000 g for 10 min. A half-volume of HCP is added to the supernatant fluid, shaken vigorously to obtain a homogeneous emulsion, and recentrifuged as above. Another half-volume of HCP is added to the supernatant and the procedure is repeated. HCP absorbs TMV and removes it from the preparations. Total removal of TMV is ascertained by always setting aside samples for infectivity tests. HCP also absorbs 80-95% of the homogenate protein, which results in 5- to 20-fold purification. However, since this is the first fraction devoid of TMV, it is also the first one for which antiviral activity can be estimated and is designated "crude AVF." Crude AVF is dialyzed against H20 and lyophilized. It is stable in this form in a desiccator at room temperature for many months. Step 2: Chromatography on DEAE-Cellulose. A column of DEAEcellulose (Whatman DE-23 or DE-52, 0.6 x 15 cm) is equilibrated with the standard buffer. Crude AVF (200 mg protein) is dissolved in the buffer and applied to the column. A gradient of 0-0.75 M NaC1 in the standard buffer is applied. Fractions, diluted with the standard buffer (1 : 1000), are tested for antiviral activity. Mock AVF preparations are similarly prepared and assayed (Fig. 3). AVF is eluted from DEAE-cellulose in a very peculiar position for a protein (0.48-0.55 M NaC1) probably because it is phosphorylated. Indeed, only 2% of the protein is eluted at the AVF position. At this stage, however, the purification factor is far greater than expected from the amount of protein removed (see the table), indicating that inhibitors to AVF activity are apparently removed.
E
,,-°
1.4
I (
0.02-04M NaCI
,.0I-
N o.6F v
I
<'P-04-06M NaCI--> > ?'ill"
-!f 1 '.,If16O 'oL
i 5
I0 15 FRACTION NUMBER
1 20
FIG. 3. Elution pattern of AVF from DEAE cellulose. Absorbancy of proteins from a mock preparation (~) and from AVF preparation (4.). Antiviral activity of AVF (A). Data from Antignus e t a l . ~
742
[97]
INTERFERON AND PLANT CELLS SUMMARY OF PURIFICATION OF A V F
Fraction Leaf homogenate Crude AVF DEAE-AVF Con A-AVF HPLC-AVF (Fraction No. 39)
Total protein (txg)
Dilution end-point of A V F activity (pg protein/ml)
Estimated purification factor
200,000 30,000 600 80 2.4
-150,000 6 0.8 0.24
-1 25,000 187,500 625,000
In routine preparations, AVF is eluted from DEAE-cellulose in a stepwise manner between 0.45 and 0.60 M NaCI. It is dialyzed against H20, lyophilized, and stored as crude AVF, designated D E A E - A V F . Step 3: Affinity Chromatography on Concanavalin A-Sepharose. A column (0.6 × 10 cm) of concanavalin A-Sepharose (Pharmacia) is equilibrated with the Con A buffer. D E A E - A V F , 10-50 mg protein in the same buffer, is applied to the column and washed with 30 ml of the Con A buffer. A V F is eluted with 5% a-methyl-D-mannoside (Sigma) in the above buffer. A minute quantity of concanavalin A is constantly leaking from the column, and, since there is very little AVF at this stage, con-
6ofA OLD~
I
t
t
t
2O 0
I0
20 30 40 50 60 70 FRACTION NUMBER
FIG. 4. Elution patterns o f antiviral activity from r e v e r s e - p h a s e H P L C (see text for details). Disks o f TMV-infected tobacco leaves were i m m e r s e d in the standard buffer containing the various c o l u m n fractions for 72 hr at which time the virus content was determined by E L I S A . (A) Fractions of m o c k A V F preparation. (B) Fractions of A V F preparation. The T M V c o n t e n t of buffer-treated leaf disks fell between the dashed lines. The antiviral activity of the A V F fractions a c c u m u l a t e d in a wide zone, and was especially strong in fraction No. 39.
[97]
PLANT
ANTIVIRAL
140
z
'
I
t
I
PROTEIN
I
'
I
743
I
r
120
I,LI
"
IO0
Ow Q'J
80
•-,1 i.L ILl
-
6O enW
~o_
40
W _
,
TIME AFTER INOCULATIONlhr)
F1G. 5. TMV growth curve of control leaf disks and of disks treated with fraction No. 39 of Fig. 4. TMV in leaf disks was estimated by the infectivity of the disk homogenates. (0) Fraction No. 39; (O) control.
canavalin A contamination is considerable. The preparation is therefore dialyzed against the standard buffer and reapplied to DEAE-cellulose. Concanavalin A is eluted from the column with 0.45 M NaC1 and AVF is then eluted with 0.60 M NaCI. The AVF fraction (designated Con A AVF) is dialyzed against water, lyophilized, and stored in a desiccator at room temperature.
Purification by HPLC This is still at the experimental stage and should be so regarded. Crude AVF is dissolved in the HPLC buffer, loaded on a reverse-phase column (octyl silica, Merck RP-8, 10 /xm), and chromatographed with a linear gradient (0-40%) of n-propanol as described for human leukocyte interferon by Rubinstein and Pestka. 27 A great number of fractions eluted in the area exhibiting antiviral activity (Fig. 4). The most active fraction (No. 39) was further tested for antiviral activity (Fig. 5). Since about 10 7 cells are in a leaf disk and the molecular weight of AVF is estimated to be 22,000 (see below), it seems that less than 1 AVF molecule per cell is sufficient to confer protection from TMV.
~7 M. Rubinstein and S. Pestka, this series, Vol. 78, p. 464.
744
INTERFERON
AND PLANT
CELLS
[98]
Properties
Stability AVF, at every stage, can be stored, dry, at room temperature for months without loss of activity. Con A - A V F remains active after being dialyzed at pH 2 for 48 hr and then back to pH 7.6. D E A E - A V F and Con A-AVF remain active in 0.1% SDS but must be diluted for the antiviral assay. AVF can be eluted in an active form from SDS-polyacrylamide gels.
Purity D E A E - A V F appears as a broad zone on polyacrylamide gel electrophoresis indicating heterogeneity of the active molecules. At least 20 protein bands, on a background of a smear, are observed upon staining. The expansion of the stained area beyond the active zone indicates that AVF is far from pure at this stage. Con A-AVF upon electrophoresis also appears as a zone of activity, but in this case the stained area is superimposed on that of antiviral activity. The center of the AVF zone migrates as a protein with a MW of 22,000. Fraction No. 39 of Fig. 4 migrates as a highly active homogeneous protein of MW 21-22,000 on SDS-polyacrylamide gel electrophoresis.
[98] A s s a y o f E f f e c t o f H u m a n I n t e r f e r o n s on Tobacco Protoplasts
By ILAN SELA Human interferons were found to protect tobacco leaf disks from TMV I infection2 and induce the polymerization of ATP to antivirally active oligonucleotides. 3,4 The interferon (and AVFS)-induced plant nucleotides are not identical to the animal 2,5-An, 4'6 but the latter and many of its derivatives inhibit TMV multiplication.7'8 i TMV; tobacco mosaic virus. 2 p. Orchansky, M. Rubinstein, 3 M. Reichman, Y. Devash, R. 4 y . Devash, I. Sela, and R. J. ~ AVF: TMV-induced antiviral
and 1. Sela, Proc. Natl. Acad. Sci. U.S.A. 79, 2279 (1982). J. Suhadolnik, and 1. Sela, Virology 128, 240 (1983), Suhadolnik, this volume [99]. factor from Nicotiana glutinosa.
METHODS IN ENZYMOLOGY, VOI.. 119
Copyright (,) 1986 by Academic Press, Inc. All righls of reproduction in any form reserved.
INTERFERON AND PLANT CELLS
[97]
Further Purification and Molecular Weight Estimation of IVR Further purification and estimation of the molecular weight were done by gel filtration through a Sephadex G-75 column. 7 A lyophilized IVR preparation obtained from 3 × 107 inoculated protoplasts was dissolved in I ml of 0.1 M phosphate buffer, pH 6.0 (OD280 = 2), placed on a precalibrated Sephadex G-75 column (33 × 2.3 cm), and eluted with the same buffer at a flow rate of 100 ml/hr. A control preparation obtained from a similar number of noninoculated protoplasts with the OD280 adjusted to 2 was passed through the same column in a similar way. Inhibitory potency of each third fraction, in comparison with the respective control fraction, was tested as before on inoculated Samsunn NN protoplasts. Peaks of activity were eluted at two positions, equivalent to molecular weights of 26,000 and 57,000. Characteristics of IVR IVR was stable in pH 2.5 and the antiviral activity was conserved after dialysis against 0.05 M phosphate buffer, pH 7.5, or against VIM. IVR was inactivated completely or its activity markedly reduced by treatment with trypsin or chymotrypsin, but not with ribonuclease? IVR was found to be thermolabile and was inactivated by heating at 60° for 10 min. These data suggest that IVR is of a proteineous nature. 7 j. R. Whitaker, Anal. Chem. 35, 1950 (1963).
[97] P r e p a r a t i o n a n d M e a s u r e m e n t o f a n A n t i v i r a l P r o t e i n F o u n d in T o b a c c o Cells a f t e r I n f e c t i o n w i t h Tobacco Mosaic Virus
By
ILAN SELA
AVF (antiviral factor) is a cumulative name given by Sela and Applebaum ~ to what turned out to be a group of closely related proteinaceous plant substances with antiviral activity. AVF, or its precursors, also present in noninfected tobacco leaves, is easily stimulated by various i 1. Sela and S. W. Applebaum, Virology 17, 543 (1962).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 119
Copyright (t) 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
[97]
PLANT ANTIVIRAL PROTEIN
735
agents, 2 notably poly(I) • poly(C) 3 and especially TMV. 4 AVF stimulation is most pronounced following TMV infection of plants carrying the Ngene, which is responsible for localizing TMV infection in these plants 5,6 (for discussion of the N-gene see Sela2). A substance similar to AVF was later described in N-gene-carrying tobacco mesophyll protoplasts. 7,8 AVF, or some of it, is a phosphorylated glycoprotein. 9,~° Its activity in tissues, estimated at 0.1-10 molecules per cell, triggers some primary reactions whose products communicated to neighboring cells impose an "antiviral state."2 A major activity of AVF (and human leukocyte interferon) in tobacco cells is the stimulation of a synthetase activity which polymerizes ATP to oligoadenylates in the presence of dsRNA. 11 The plant oligonucleotides though antivirally active ~2 are not identical with 2,5-A,. 13,14AVF resembles interferon ~5which was indeed found to protect plant tissues from T M V , 16'17 as well as 2,5-A~ 18 and its derivatives. 19 Assaying Antiviral Activity in Plants General Comments on Quantitation
Measuring infectivity is a prerequisite for measuring antiviral activity. The lack of clonal cell lines in plants makes it impossible to develop a truly quantitative assay. Although a newly developed assay with protoplasts from standard tobacco cell suspensions, 2° is, so far, the best assay
2 I. Sela, Adv. Virus Res. 26, 201 (1981). 3 0 . Gat-Edelbaum, A. Altman, and 1. Sela, J. Gen. Virol. 64, 211 (1983). 4 TMV: tobacco mosaic virus. Y. Antignus. i. Sela, and I. Harpaz, J. Gen. Virol. 35, 107 (1977). ~' 1. Sela, A. Hauscbner, and R. Mozes, Virology 89, 1 (1978). 7 G. Loebenstein and A. Gera, Virology 114, 132 (1981), A. Gera, S. Spiegel, and G. Loebenstein, this volume [96]. Y. Antignus, 1. Sela, A. Hauschner, and !. Harpaz, Physiol. Plant Pathol. 6, 159 (1975). l0 R. Mozes, Y. Antignus, I. Sela, and I. Harpaz, J. Gen. Virol. 38, 241 (1978). ~t y . Devash, A. Hauschner, I. Sela, and K. ChakraburUy, Virology 111, 103 (1981). ~2 M. Reichman, Y. Devash, R. J. Suhadolnik, and I. Sela, Virology 128, 240 (1983). ~ Y. Devash, M. Reichman, 1. Sela, and R. J. Suhadolnik, Biochemist O, 24, 593 (1985). 14 y . Devash, I. Sela, and R. J. Suhadolnik, this volume [99]. is 1. Sela, Perspect. Virol. 11, 129 (1981). ~ P. Orchansky, M. Rubinstein, and I. Sela, Proc. Natl. Acad. Sci. U.S.A. 79, 2278 (1982). ~7 N. Rosenberg, M. Reichman, A. Gera, and 1. Sela, Virology 140, 173 (1985). t8 y . Devash, S. Biggs, and I. Sela, Science 216, 1415 (1982). ~9 y . Devash, A. Gera, M. Reichman, W. Pfleiderer, R. Charubala, I. Sela, and R. J. Suhadolnik, J. Biol. Chem. 259, 3482 (1984). -"~ I. Sela, M. Reichman, and A. Weissbach, Phytopathology 74, 385 (1984).
736
INTERFERON AND PLANT CELLS
[97]
for reliable quantitation of infection, it should still be considered as no more than an estimate. Measuring the antiviral activity of AVF and interferon is further complicated by the odd dose-response of tobacco tissues and cells. Interferon protects tobacco cells at a very low dosage, but its antiviral activity diminishes gradually with increase in interferon concentration. 17 With AVF at least two waves of antiviral activity are usually observed upon dilution.
Infectivity Assay: The Local Lesion Test Some viruses react with their host plants by producing local lesions (mostly necrotic) on the infected leaves. The number of local lesions is proportional to virus concentration, but, since leaves vary considerably in their response to viral infection, it is impossible to draw an accurate standard curve. In order to overcome this inherent shortcoming, at least partially, we usually combine the half-leaf test with one involving a dilution end-point. Purified TMV (prepared according to Devash et al. j~) is added to AVF or mock AVF 21 solutions to a final concentration of 5/~g TMV per ml 0.01 M sodium phosphate, pH 7.6, and carborundum (400600 mesh) is added to a concentration of 0.1 g/ml. The content of the tube is stirred with a sterile painter's brush which is then squeezed against the inner tube wall to remove excess liquid. Half-leaves of Datura stramenium L. are then inoculated by gently moving the brush from tip to petiol, creating 10-mm-wide inoculated strips. Control and AVF solutions are applied to opposite halves of the same leaves (Fig. I). A series of AVF dilutions to about 0.01 pg protein per ml are thus tested (the TMV concentration remains constant), each dilution being applied to 12 half-leaves. The plants are kept at 24 ° under constant illumination and local lesions are counted 3-4 days later. Activity at each dilution is expressed as a percentage of protection of the AVF-treated half-leaves compared to the control half-leaves.
The L e a f Disk Test In this test the viral content of leaf disks is determined either by the above described infectivity tests of disk homogenates, or, more reliably, by the enzyme-linked immunosorbent assay (ELISA). ~2 2i Mock AVF: A fraction prepared similarly to the particular AVF fraction but from noninfected plants. 22 M. F. Clark and A. H. Adams, J. Gen. Virol. 34, 475 (1977).
[97]
PLANT ANTIVIRAL PROTEIN
737
FIG. I. Half-leaf test. Leaves of D. stramonium were inoculated with 5 /zl/ml TMV containing 100 ng protein of mock DEAE-AVF preparation (left half) and 100 ng protein of DEAE-AVF (right half).
ELISA Reagents
PBS-Tween: 0.5 ml T w e e n 20 (Sigma), 0.2 g NaN3 in 1 liter PBS Coating buffer: 1.59 g Na2CO3, 2.93 g NaHCO3, 0.2 g NaN3 in 1 liter H20 P B S - T w e e n - P V P : 20 g PVP-40 (polyvinylpyrrolidone, Sigma) in 1 liter P B S - T w e e n P B S - T w e e n - P V P - B S A : 0.2% bovine serum albumin (BSA) in PSBTween-PVP Substrate buffer: 97 ml diethanolamine, 800 ml H20, 0.2 g NAN3. Titrate with HCI to pH 9.8 and bring to 1 liter with H20 Substrate solution: p-nitrophenyl phosphate. Keep solid at - 2 0 °. Prepare a fresh solution of 0.1 mg/ml in substrate buffer before each use Microplates: Dynatech 129B or Nunc Immunoplates
738
INTERFERON AND PLANT CELLS
[97]
ELISA Procedure The y-globulin fraction of chicken and rabbit sera immunized against TMV is prepared according to Clark and Adams z2 and stored at 1 mg/ml at - 2 0 °. To each well of a microtiter plate 200 /xl of chicken y-globulin, diluted to 1/zg/ml in coating buffer, is added and incubated for 3 hr at 30° or overnight at 4°. The wells are thoroughly washed (3 times) with PBSTween and 200/zl samples of TMV or disk homogenates (both prepared in PBS-Tween-PVP) are added to every well and incubated as above. Following washing, rabbit y-globulin (1 ~g/ml in PBS-Tween-PVP) is added to the wells and incubated as above. Commercially available goat antirabbit IgG (conjugated to alkaline phosphatase and diluted 1 : 1000 in PBSTween-PVP-BSA) is added next. Following incubation and washing as above, 250 ~1 of substrate solution is added and incubated at room temperature. Absorption at 410 nm is determined in a Microelissa Autoreader (Dynatech MR-580). Since a 96-well plate is read in less than a minute, the reaction is not stopped and readings are taken at 10 min intervals until 1 /zg/ml TMV yields an absorption of about 0.8. TMV standards for calibration curves are included in every plate, and every sample is applied in triplicate. Absorbance is linear with TMV concentration bewteen approximately 10 and 1000 ng/ml. TMV content is expressed as ng/disk. Since the TMV concentration must fall in the linear range, serial dilutions are made from each disk homogenate.
Antiviral Assay by ELISA Leaves of tobacco var. " S a m s u n " are dusted with carborundum and are inoculated with TMV (1/xg/ml in 0.01 M sodium phosphate, pH 7.6). After extensive washing with water, leaf disks (6 or 8 mm in diameter) are punched out with a paper-punch into a beaker containing the standard buffer. Twenty disks are collected at random from the common pool and placed in Petri dishes floating on 20 ml of the above buffer. Some plates contain disks from noninfected leaves, some contain untreated inoculated disks, and the rest contain the various materials to be tested for antiviral activity (added to the disks within 1 hr after inoculation). Plates are incubated at 24° under constant illumination. The disks are then frozen at - 2 0 °, homogenated with a Teflon-coated, motor-driven homogenizer, and their TMV content is determined by ELISA. TMV is first detected (in homogenate of 20 disks) 36 hr after inoculation, and increases rapidly between 48 and 96 hr after inoculation. It is better to follow the TMV growth curve in control and tested solutions, but since, in many cases, this is too elaborate a task, a single time-point between 60 and 80 hr is
[97]
PLANT ANTIVIRAL PROTEIN
739
D I L U T I O N OF CELL LYSATES
TREATMENT 1
1:2
1:4
1:8
1:16
1:32
FIG. 2. Dot-blot assay. TMV-inoculated protoplasts were treated with interferon (IFNaA; 0.1 units/ml) and with D E A E - A V F (60 ng protein/ml). Cell lysates were serially diluted (from left to right), spotted onto nitrocellulose, and hybridized as described in the text.
chosen. The homogenate of 20 disks, 72 hr after inoculation, should be diluted 1 : 10-1 : 100 for ELISA.
Antiviral Assay by Dot-Blot Test This test, developed in order to detect TMV-RNA in crude leaf and protoplast extracts, is about 100 times more sensitive than the ELISA. 2° This assay is based on a specific hybridization between radioactive cDNA (reverse-transcribed from TMV-RNA) and the RNA in the extracts and measures TMV-RNA accumulation. The detailed procedure is described elsewhere. 23 AVF and human interferon suppress TMV-RNA replication. The availability of highly purified interferon enables the expression of its activity in the protoplast system in terms of molecules per cell. By comparing cruder AVF preparations to interferon in the same experiment, interferon units could be assigned to AVF (Fig. 2). Purification AVF elutes as a broad peak of antiviral activity upon DEAE-cellulose chromatography and HPLC. After electrophoresis it appears as a broad z3 1. Sela, this volume [98].
740
INTERFERON AND PLANT CELLS
[97]
zone rather than a sharp band. The better resolving HPLC procedure exhibits, within the area of AVF activity, a number of protein peaks, some of which appear homogeneous on electrophoresis. Hence, it seems that AVF is a family of closely related molecules. The AVF preparations described herein cannot, therefore, be regarded as homogeneous. One HPLC fraction, which was studied a number of times was found to be homogeneous; however, it is premature to draw any definitive conclusions in this respect.
Reagents Standard buffer: 0.01 M sodium phosphate, pH 7.6 HCP (hydrated calcium phosphate24): a volume of 0.1 M Na2HPO4 is added to the same volume of 0. I M CaCI2. A precipitate is formed and washed 10 times with HzO by decantation. HCP is centrifuged prior to its addition to plant material to remove excess liquid Con A buffer: 0.1 M sodium acetate, pH 6.0; 1 mM MnCI2; 1 mM MgCI2; 1 mM CaCI2 HPLC buffer: 1 M pyridine, 2 M formic acid, pH 4.0
Protein Determinations A 50 /zl sample of an AVF preparation is mixed with 50 /xl of a Coomassie blue reagent 25,26 in an ELISA microtiter plate and the developed color is read at 570 nm in the automated ELISA reader. A calibration curve of known BSA concentrations is read in every plate. Green plant homogenate is precipitated with 7% perchloric acid and then redissolved in 0.1 N NaOH for protein determination. Dilutions (at least 1 : 10 in H20) of this basic solution can be added to the reagent and read quantitatively.
Plant Material Nicotiana glutinosa plants (carrying the N-gene) are grown in an insect-proof green house at 24 -+ 2° under constant illumination. At the stage of 4-6 fully expanded leaves and following dusting with carborundum, the leaves are rubbed gently with TMV (5 ~g/ml in the standard buffer). Leaves are harvested 2 days later when distinct local lesions appear but are not yet fully expanded.
24 R. W. Fulton, Virology 9, 522 (1959). 25 j. j. S e d m a k and S. E. Grossberg, Anal. Biochem. 79, 544 (1977). 26 Commercial kit with protocol from Bio-Rad Laboratories.
[97]
PLANT ANTIVIRAL PROTEIN
741
Procedure Step 1: Preparation of Crude AVF. The leaves are homogenized in a blender in the standard buffer at a ratio of 1:1 (w/v). The homogenate is squeezed through 3 layers of cheesecloth and centrifuged at 5000 g for 10 min. A half-volume of HCP is added to the supernatant fluid, shaken vigorously to obtain a homogeneous emulsion, and recentrifuged as above. Another half-volume of HCP is added to the supernatant and the procedure is repeated. HCP absorbs TMV and removes it from the preparations. Total removal of TMV is ascertained by always setting aside samples for infectivity tests. HCP also absorbs 80-95% of the homogenate protein, which results in 5- to 20-fold purification. However, since this is the first fraction devoid of TMV, it is also the first one for which antiviral activity can be estimated and is designated "crude AVF." Crude AVF is dialyzed against H20 and lyophilized. It is stable in this form in a desiccator at room temperature for many months. Step 2: Chromatography on DEAE-Cellulose. A column of DEAEcellulose (Whatman DE-23 or DE-52, 0.6 x 15 cm) is equilibrated with the standard buffer. Crude AVF (200 mg protein) is dissolved in the buffer and applied to the column. A gradient of 0-0.75 M NaC1 in the standard buffer is applied. Fractions, diluted with the standard buffer (1 : 1000), are tested for antiviral activity. Mock AVF preparations are similarly prepared and assayed (Fig. 3). AVF is eluted from DEAE-cellulose in a very peculiar position for a protein (0.48-0.55 M NaC1) probably because it is phosphorylated. Indeed, only 2% of the protein is eluted at the AVF position. At this stage, however, the purification factor is far greater than expected from the amount of protein removed (see the table), indicating that inhibitors to AVF activity are apparently removed.
E
,,-°
1.4
I (
0.02-04M NaCI
,.0I-
N o.6F v
I
<'P-04-06M NaCI--> > ?'ill"
-!f 1 '.,If16O 'oL
i 5
I0 15 FRACTION NUMBER
1 20
FIG. 3. Elution pattern of AVF from DEAE cellulose. Absorbancy of proteins from a mock preparation (~) and from AVF preparation (4.). Antiviral activity of AVF (A). Data from Antignus e t a l . ~
742
[97]
INTERFERON AND PLANT CELLS SUMMARY OF PURIFICATION OF A V F
Fraction Leaf homogenate Crude AVF DEAE-AVF Con A-AVF HPLC-AVF (Fraction No. 39)
Total protein (txg)
Dilution end-point of A V F activity (pg protein/ml)
Estimated purification factor
200,000 30,000 600 80 2.4
-150,000 6 0.8 0.24
-1 25,000 187,500 625,000
In routine preparations, AVF is eluted from DEAE-cellulose in a stepwise manner between 0.45 and 0.60 M NaCI. It is dialyzed against H20, lyophilized, and stored as crude AVF, designated D E A E - A V F . Step 3: Affinity Chromatography on Concanavalin A-Sepharose. A column (0.6 × 10 cm) of concanavalin A-Sepharose (Pharmacia) is equilibrated with the Con A buffer. D E A E - A V F , 10-50 mg protein in the same buffer, is applied to the column and washed with 30 ml of the Con A buffer. A V F is eluted with 5% a-methyl-D-mannoside (Sigma) in the above buffer. A minute quantity of concanavalin A is constantly leaking from the column, and, since there is very little AVF at this stage, con-
6ofA OLD~
I
t
t
t
2O 0
I0
20 30 40 50 60 70 FRACTION NUMBER
FIG. 4. Elution patterns o f antiviral activity from r e v e r s e - p h a s e H P L C (see text for details). Disks o f TMV-infected tobacco leaves were i m m e r s e d in the standard buffer containing the various c o l u m n fractions for 72 hr at which time the virus content was determined by E L I S A . (A) Fractions of m o c k A V F preparation. (B) Fractions of A V F preparation. The T M V c o n t e n t of buffer-treated leaf disks fell between the dashed lines. The antiviral activity of the A V F fractions a c c u m u l a t e d in a wide zone, and was especially strong in fraction No. 39.
[97]
PLANT
ANTIVIRAL
140
z
'
I
t
I
PROTEIN
I
'
I
743
I
r
120
I,LI
"
IO0
Ow Q'J
80
•-,1 i.L ILl
-
6O enW
~o_
40
W _
,
TIME AFTER INOCULATIONlhr)
F1G. 5. TMV growth curve of control leaf disks and of disks treated with fraction No. 39 of Fig. 4. TMV in leaf disks was estimated by the infectivity of the disk homogenates. (0) Fraction No. 39; (O) control.
canavalin A contamination is considerable. The preparation is therefore dialyzed against the standard buffer and reapplied to DEAE-cellulose. Concanavalin A is eluted from the column with 0.45 M NaC1 and AVF is then eluted with 0.60 M NaCI. The AVF fraction (designated Con A AVF) is dialyzed against water, lyophilized, and stored in a desiccator at room temperature.
Purification by HPLC This is still at the experimental stage and should be so regarded. Crude AVF is dissolved in the HPLC buffer, loaded on a reverse-phase column (octyl silica, Merck RP-8, 10 /xm), and chromatographed with a linear gradient (0-40%) of n-propanol as described for human leukocyte interferon by Rubinstein and Pestka. 27 A great number of fractions eluted in the area exhibiting antiviral activity (Fig. 4). The most active fraction (No. 39) was further tested for antiviral activity (Fig. 5). Since about 10 7 cells are in a leaf disk and the molecular weight of AVF is estimated to be 22,000 (see below), it seems that less than 1 AVF molecule per cell is sufficient to confer protection from TMV.
~7 M. Rubinstein and S. Pestka, this series, Vol. 78, p. 464.
744
INTERFERON
AND PLANT
CELLS
[98]
Properties
Stability AVF, at every stage, can be stored, dry, at room temperature for months without loss of activity. Con A - A V F remains active after being dialyzed at pH 2 for 48 hr and then back to pH 7.6. D E A E - A V F and Con A-AVF remain active in 0.1% SDS but must be diluted for the antiviral assay. AVF can be eluted in an active form from SDS-polyacrylamide gels.
Purity D E A E - A V F appears as a broad zone on polyacrylamide gel electrophoresis indicating heterogeneity of the active molecules. At least 20 protein bands, on a background of a smear, are observed upon staining. The expansion of the stained area beyond the active zone indicates that AVF is far from pure at this stage. Con A-AVF upon electrophoresis also appears as a zone of activity, but in this case the stained area is superimposed on that of antiviral activity. The center of the AVF zone migrates as a protein with a MW of 22,000. Fraction No. 39 of Fig. 4 migrates as a highly active homogeneous protein of MW 21-22,000 on SDS-polyacrylamide gel electrophoresis.
[98] A s s a y o f E f f e c t o f H u m a n I n t e r f e r o n s on Tobacco Protoplasts
By ILAN SELA Human interferons were found to protect tobacco leaf disks from TMV I infection2 and induce the polymerization of ATP to antivirally active oligonucleotides. 3,4 The interferon (and AVFS)-induced plant nucleotides are not identical to the animal 2,5-An, 4'6 but the latter and many of its derivatives inhibit TMV multiplication.7'8 i TMV; tobacco mosaic virus. 2 p. Orchansky, M. Rubinstein, 3 M. Reichman, Y. Devash, R. 4 y . Devash, I. Sela, and R. J. ~ AVF: TMV-induced antiviral
and 1. Sela, Proc. Natl. Acad. Sci. U.S.A. 79, 2279 (1982). J. Suhadolnik, and 1. Sela, Virology 128, 240 (1983), Suhadolnik, this volume [99]. factor from Nicotiana glutinosa.
METHODS IN ENZYMOLOGY, VOI.. 119
Copyright (,) 1986 by Academic Press, Inc. All righls of reproduction in any form reserved.