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Antiviral activity of natural and recombinant human leukocyte interferons in tobacco protoplasts
VIROLOGY
140.173-178
(1985)
SHORT COMMUNICATIONS Antiviral Activity of Natural and Recombinant Human Leukocyte lnterferons in Tobacco Protoplasts N. ROSENBERG,*
M. REICHMAN,**~
*Virus Laboratory, The Hebrew University, Faculty Laboratory, Agricultuml Research Organization, Received January
A. GERA,t
AND
I. SELA*,~~’
of Agriculture,
Rehuvot 76100, Israel, and f Virus The Volcani Center, Bet Do,gan, 50250, Israel
25, 198& accepted June SO, 1984
Several purified species of human leukocyte interferon, including recombinant interferons, inhibit the multiplication of tobacco mosaic virus (TMV) in tobacco protoplasts derived from various cultivars. Viral RNA accumulation was determined by dot-blot hybridization to specific cDNA probes, and virus antigen was determined serologically. Interferon apparently inhibited both TMV-RNA replication and its expression into coat protein. However, these effects were of limited duration. Maximum effect was obtained when interferon was applied to the cells either prior to inoculation or within the first hour after inoculation. Antibodies to interferon abolished its antiviral activity in protoplasts. Tobacco protoplasts were about 1000 times more responsive to interferon than the reference animal viral-cell system and showed an “antiviral state” at a ratio of 1 molecule of interferon per Cell. 0 1985 Academic Press, Inc.
Interferons are antiviral proteins released by animal cells in response to virus infection and other stimuli (1). An antiviral factor (AVF) with properties similar to interferon occurs in virus-infected plants (reviewed by Sela; 2). A similar factor, termed IVR, is released into the medium when certain tobacco protoplasts (Samsun NN) are inoculated with tobacco mosaic virus (TMV; 3). AVF induces, in plants, some activities which resemble interferon-stimulated activities in animal tissues (4), in particular a double-stranded RNA-dependent ATP polymerization (5). Antiviral agents active in the animal kingdom were shown to be antivirally active in plants in two cases: Human interferons inhibited TMV multiplication in tobacco tissue (6) and 2’-5’ oligoadenylate (trimer) inhibited TMV infection (7, 8). Interferon, as well as AVF, also induces
the synthesis of nucleotides in plants which are antivirally active (9) and inhibit protein synthesis in rabbit reticulocyte lysates. The plant oligoadenylates did not activate an endonuclease nor compete with 2’-5’ oligoadenylate in a radiobinding assay (16). This explains why Cayley et al. (10) could not detect these oligonucleotide by radiobinding assays. A number of derivatives of 2’-5’ oligoadenylated “cores” were also found to inhibit TMV infection (11). In addition to the previously reported effect of interferon on virus production and coat protein synthesis, this paper reports also on interferon-mediated inhibition of TMV-RNA accumulation. The tobacco protoplast system used in this study allows kinetic studies as well as an investigation of the various parameters affecting interferon activity. Tobacco cells, protoplast preparation, and inoculation, and dot-blot assays for TMV-RNA were carried out essentially as described by Sela et al. (LZ), who recently reported a procedure for detecting TMV-RNA at the picogram level. Proto-
i Author to whom requests for reprints should be addressed. a Part of this work was carried out by the indicated authors at Roche Institute of Molecular Biology, Nutley, N. J. 07110.
173
0042-6822/85 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
174
COMMUNICATIONS
plasts were prepared from cultured cell suspensions of tobacco var. Wisconsin 38. Determination of TMV coat protein in protoplasts was carried out by the enzymelinked immunosorbent assay (ELISA) as described by Orchansky et aa (6). Sedimented protoplasts were diluted 1:3 in a mannitol-free ELISA sample buffer and homogenized. A standard TMV dilution curve was included in every experiment. TMV-RNA accumulation in protoplasts was followed by removing aliquots at various time intervals after inoculation with TMV. Protoplasts were lysed, dotted on nitrocellulose, and hybridized with cDNA probes specific to TMV-RNA as described by Sela et al. (1.2). After autoradiography films were scanned in a densitometer (Cliniscan, Helena Laboratories) or alternatively, the dots were cut out and their radioactivity was measured in a liquid scintillation spectrometer.
Interferons
and interferon
treatments.
All interferon preparations were highly purified: (i) subspecies 73 (2.4 X lo8 units/ mg) of Rubinstein et al. (13) was obtained from M. Rubinstein, Weizmann Institute of Science, Rehovot, Israel. (ii) Recombinant human leukocyte interferon preparation IFN-aD (2.2 X lo8 units/mg), equivalent to the above-mentioned natural interferon 73, was obtained from S. Pestka, Roche Institute of Molecular Biology, Nutley, New Jersey. (iii) Recombinant interferon preparation IFN-aA (2.9 X lOa units/ mg), equivalent to the natural interferon cr2/31, was obtained from the Department of Molecular Genetics, Roche Research Center, Nutley, New Jersey. For information about classification and terminology of human leukocyte interferons see Pestka (1). The recombinant interferon preparations were obtained as described by Staehelin et al. (14) and Rehberg et
aL (15). Interferon was diluted to the desired concentration with the Murashige and Skoog medium as specified elsewhere (12) containing 12% mannitol and 1% fetal calf serum (FCS). Interferon at lo-fold the desired concentration (0.1 vol) was added to a 0.9 vol of protoplasts, so that the
final FCS concentration was always 0.1%. Higher concentrations of FCS caused the appearance of filamentous structures and protoplasts aggregation. A similar interferonless solution containing mannitol and FCS served as a mock interferon preparation. A polyclonal antibody preparation against interferon IFN-aA was obtained from S. Pestka. One milliliter of this preparation was sufficient to neutralize 50% of the activity of 1.7 X lo6 interferon units. In order to test the effect of these antibodies on interferon activity in tobacco protoplasts, 8500 antibody units were incubated with 1000 interferon units for 15 min at 37”. The interferon was then further diluted (103-106-fold) and applied to the protoplasts. Tobacco protoplasts were prepared, as described, from cell suspensions (Wisconsin 38) or leaf mesophyll (“Samsun”; “Samsun NN”). The Wisconsin 38 protoplasts were maintained at lo6 cells/ml and their virus content was determined by ELISA ‘72 hr after TMV inoculation. The mesophyll protoplasts were shaken as suspensions and their virus titers were determined by both ELISA and infectivity 48 hr postinoculation. Pretreatment with all three subspecies of leukocyte interferon, added to a final concentration of 1 unit/ml, protected three kinds of protoplasts from subsequent TMV infection (Table 1). Some protection was observed even after brief exposure to interferon (15-30 min), but the antiviral effect became substantial only after l-3 hr. When added 3 hr post-TMV inoculation, interferon had a much lesser effect, requiring a longer time in order to protect against viral infection (data not shown). In practice, interferon was found to confer maximum protection also when added within 1 hr after TMV inoculation (Fig. 2). In order to investigate the effect of interferon on TMV-RNA accumulation, protoplasts were pretreated with interferon IFN-aA (1 unit/ml, 3 hr) and then washed and inoculated with TMV. At various times postinoculation, aliquots were
-
-
381 (0)f
380 (0.3)
241 (38)
192 (51)
89 (77)
69 (82)
54 (86) 48 (88)
-
No additions
Mock, 12 hr
15 min
30 min
1 hr
3 hr
6 hr
12 hr
24 hr
TMV/lO”
Nanograms
-
of Nicotiana
30 (87)
-
38 (83) -
-
81 (79) 40 (90) 30 (92)
230 (0) 124 (46) -
245 (37) 176 (55)
-
-
glutinosa
and the number
30 (86)
-
31 (85)
211 (0) 110 (48) -
-
INFECTION
3 (92)
-
of 12 leaves).
2 (86)
-
5 (64) -
-
7 (81)
14 (0) 8 (43) -
-
Samsun NNd treated with y3
37 (0) 18 (51) -
-
Samsund treated with y3
Number of local lesions caused by protoplasts homogenatese
ON TMV
of lesions was counted (average
Samsun NNd treated with y3
protoplasts”
LEUKOCYTE INTERFERONS OF TOBACCO PROTOPLASTS
Samsund treated with y3
VARIANTS
IFN-aD
38’
1
WITH VARIOUS TYPES OF HUMAN IN SEVERAL
’ 1 unit/ml of the indicated type of interferon. *Determined by ELISA. ‘Prepared from cell suspensions. d Mesophyll protoplasts. e Protoplast homogenates (106/ml) were applied to half-leaves ‘Numbers in parentheses are percentage protection values.
30 (92) -
54 (86)
90 (77)
252 (34)
381 (19)
IFNCXA
Wisconsin
Treated with interferon Y3
Time of exposure to interferon” (prior to inoculation)
THE EFFECT OF PRETREATMENTS
TABLE
SHORT
176
COMMUNICATIONS
a Protoplasts (Wisconsin 38) were pretreated for 3 hr with 1 unit/ml of the interferon subspecies IFNolA, washed, and then inoculated with TMV.
lysed, and samples, equivalent to 25,000 cells per dot, were applied to nitrocellulose and hybridized with a cDNA probe as described. Interferon was found to be most active in preventing TMV-RNA replication in tobacco protoplasts at concentrations of 0.01-0.1 units/ml, inhibiting about 50% of TMV-RNA replication even at a concentration of 0.001 unit/ml. Increasing the interferon concentration above 1.0 unit/ml decreased its antiviral activity, but did not abolish it (Fig. 1). Experiments, similar to the above-described dose-response assays, were carried out, but the interferon was added immediately after rather than before TMV inoculation. In addition, part of the interferon preparation (1000 units/ml) was incubated with an anti-interferon antibody, at levels sufficient to neutralize four times the interferon concentration. The antibody-treated interferon was then diluted to 0.001-l units/ml and applied to the protoplasts. Protoplasts were then lysed,
taken for either dot-blot hybridization with a cDNA probe made from TMVRNA or TMV coat protein determination by ELISA. Following interferon treatment TMVRNA accumulation was almost totally inhibited for at least 60 hr after inoculation and was then resumed at a slow rate so that at 120 hr postinfection a 60% inhibition was still observed (Table 2). TMV coat antigen accumulation, initially inhibited, resumed at a faster rate, reaching control levels 120 hr after infection. It seems that for the coat protein expression to proceed normally the TMV-RNA within the cell has to reach only a certain threshold level. All further experiments were carried using the dot-blot hybridization assay system because of its greater sensitivity. Protoplast dose-response to interferon was studied as follows: Protoplasts were pretreated with various concentrations of interferon IFN-CYA for 3 hr, and then washed and inoculated with TMV. At ‘72 hr after inoculation protoplasts were
FIG. 1. The effect of interferon dosage on TMVRNA accumulation in tobacco protoplasts. Suspension cell protoplasts were exposed to interferon (recombinant IFN-aA) for 3 hr, inoculated with TMV, incubated for 72 hr, lysed, and their nucleic acids were denatured as described in the text. Aliquots, equivalent to 25,000 cells per dot were applied to nitrocellulose and hybridized with a cDNA probe specific to TMV-RNA. The autoradiogram, presented for comparison, was scanned in a densitometer.
TABLE
2
ACCIJMULATION OF TMV-RNA (DOT HYBRIDIZATION) AND OF TMV COAT PROTEIN ANTIGEN (ELISA) IN TOBAKO PROTOPLASTS FOLLOWING INTERFERON TREATMENT”
post-TMV inoculation (hr)
Time
30
60
SO
120
Dot hybridization (cpm/dot) No additions Mock interferon Interferon
62 42 7
279 242 87
467 440 172
634 726 236
ELISA (ng/106 cells) No additions Mock interferon Interferon
10 0 0
300 278 24
446 481 341
605 640 631
SHORT
177
COMMUNICATIONS
ab
ob
ab
l b
IllTERFEROlIr~il~/mll FIG. 2. The effect of anti-interferon antibodies on interferon activity in tobacco protoplasts. Interferon, either untreated or preincubated with anti-interferon antibodies, was applied to tobacco protoplasts 20 min after inoculation. Analyses are as in Fig. 1. (ab = antibodies).
treated, blotted, and hybridized as before. It was clearly demonstrated that the antibody treatment abolished the inhibitory effect of interferon and returned TMVRNA accumulation to the level of control infections (Fig. 2). The antibody preparation at the employed dilutions did not inhibit TMV increase (data not shown). This communication presents evidence that human leukocyte interferon suppresses TMV accumulation in tobacco protoplasts in addition to the previously reported inhibition in tobacco leaf disks (6). It is also demonstrated that a number of different interferon preparations, including recombinant species, are similarly active in protoplasts deriving from three different sources. In a protoplast system, cells which had been exposed to interferon exhibited resistance to subsequent virus infection, demonstrating that an antiviral state did indeed take place. The buildup of this antiviral state commenced almost instantaneously, but 3-6 hr of exposure to interferon were required for maximum effect. In this study TMV-RNA accumulation was measured directly, adding a new parameter to the hitherto assayed infectivity and coat protein antigen accumulation. The data presented in Table 2 suggest
that TMV-RNA replication is a primary target for the antiviral activity of interferon in tobacco. The dose-response of tobacco protoplasts to interferon was similar to that reported for tobacco leaf disks (6), except that in the protoplast system interferon concentrations above l-10 units/ml exhibited less antiviral activity. The protoplast system allows accurate determination of the interferon level required for inhibiting TMV-RNA replication. Assuming a sp act of 2 X lo* units of interferon per milligram protein and a molecular weight of 20,000, only one or a few molecules of interferon seem sufficient to confer maximum protection on a tobacco cell. The various interferon preparations tested were essentially pure proteins. Therefore it is highly improbable that there was an antivirally active contaminant in the diluted preparations. The neutralization of the antiviral activity of interferon by the corresponding antibody makes any such possibility unlikely and confirms the reliability of the assay. REFERENCES 1. PESTKA, S., Arch.
(1983). 2 SELA, I., Adv.
virus
B&hem
Biophgs.
Res. 26,201-237
221,
(1981).
1-37
178
SHORT
8 LOEBENSTEIN, 4. 5. 6. 7. 8.
9. 10.
G.,
and
GERA,
A.,
Virology
COMMUNICATIONS
114,
132-139 (1981). WILLIAMS, R. B. G., and KERR, I. M., Trend-s Biochem sci 4,138-140 (1980). DEVASH, Y., HAUSCHNER, A., SELA, I., and CHAKRABUR’KTY, K., virdogy 111, 103-112 (1981). ORCHANSKY, P., RUBINSTEIN, M., and SELA, I., Proc Nat1 Ad Sci USA 79,2278-22X@ (1982). DEVASH, Y., BIGGS, S., and SELA, I., Science (Washington, D. C.) 216, 1415-1416 (1982). ZHANG, XIU-HUA, TIAN, Bu., CHEN, ZANG-GUN, and LIN XIN-WAN. National Interferon meeting of China, June 26-30, 1982. REICHMAN, M., DEVASH, Y., SUHADOLNIK, R. J., and SELA, I., Virology 128, 240-244 (1984). CAYLEY, P. J., WHITE, R. F., ANTONIW, J. F., WALESBY, N. J., and KERR, I. M., B&hem. Biophys. Res. Commun 108, 1243-1250 (1982).
11. DEVASH, Y., GERA, A., REICHMAN, M., PLEIDERER, W., CHARABIJLA, R., SELA, I., and SUHADOLNIK, R. J., J. Bid Chem 259, 3482-3486 (1984). 12. SELA,
I.,
REICHMAN,
Phytqathologg
M.,
and
74,395-389
WEISSBACH, (1984).
A.,
13. RUBINSTEIN, M., LEVY, W. P., MOSCHERA, J. A., LAI, C-Y., HERSHBERG, R. D., BARTLETT, R. T., and PESTKA, S., Arck B&hem Biophys. 210, 30’7-318 (1981). 14. STAEHELIN, T., HOBBS, D. S., KUNG, H., LAI, C-Y., and PESTKA, S., J. Bid Chem 256,97509754 (1981). 15. REHBERG, E., KELDER, PESTKA, S., J. Biol. (1982).
B.,
HOAL,
E.
G.,
and
Chem. 257, 11497-11502
16. DEVASH, Y., REICHMAN, M., SELA, I., and SUHAin press. DOLNIK, R. J. Biochemistry,
140.173-178
(1985)
SHORT COMMUNICATIONS Antiviral Activity of Natural and Recombinant Human Leukocyte lnterferons in Tobacco Protoplasts N. ROSENBERG,*
M. REICHMAN,**~
*Virus Laboratory, The Hebrew University, Faculty Laboratory, Agricultuml Research Organization, Received January
A. GERA,t
AND
I. SELA*,~~’
of Agriculture,
Rehuvot 76100, Israel, and f Virus The Volcani Center, Bet Do,gan, 50250, Israel
25, 198& accepted June SO, 1984
Several purified species of human leukocyte interferon, including recombinant interferons, inhibit the multiplication of tobacco mosaic virus (TMV) in tobacco protoplasts derived from various cultivars. Viral RNA accumulation was determined by dot-blot hybridization to specific cDNA probes, and virus antigen was determined serologically. Interferon apparently inhibited both TMV-RNA replication and its expression into coat protein. However, these effects were of limited duration. Maximum effect was obtained when interferon was applied to the cells either prior to inoculation or within the first hour after inoculation. Antibodies to interferon abolished its antiviral activity in protoplasts. Tobacco protoplasts were about 1000 times more responsive to interferon than the reference animal viral-cell system and showed an “antiviral state” at a ratio of 1 molecule of interferon per Cell. 0 1985 Academic Press, Inc.
Interferons are antiviral proteins released by animal cells in response to virus infection and other stimuli (1). An antiviral factor (AVF) with properties similar to interferon occurs in virus-infected plants (reviewed by Sela; 2). A similar factor, termed IVR, is released into the medium when certain tobacco protoplasts (Samsun NN) are inoculated with tobacco mosaic virus (TMV; 3). AVF induces, in plants, some activities which resemble interferon-stimulated activities in animal tissues (4), in particular a double-stranded RNA-dependent ATP polymerization (5). Antiviral agents active in the animal kingdom were shown to be antivirally active in plants in two cases: Human interferons inhibited TMV multiplication in tobacco tissue (6) and 2’-5’ oligoadenylate (trimer) inhibited TMV infection (7, 8). Interferon, as well as AVF, also induces
the synthesis of nucleotides in plants which are antivirally active (9) and inhibit protein synthesis in rabbit reticulocyte lysates. The plant oligoadenylates did not activate an endonuclease nor compete with 2’-5’ oligoadenylate in a radiobinding assay (16). This explains why Cayley et al. (10) could not detect these oligonucleotide by radiobinding assays. A number of derivatives of 2’-5’ oligoadenylated “cores” were also found to inhibit TMV infection (11). In addition to the previously reported effect of interferon on virus production and coat protein synthesis, this paper reports also on interferon-mediated inhibition of TMV-RNA accumulation. The tobacco protoplast system used in this study allows kinetic studies as well as an investigation of the various parameters affecting interferon activity. Tobacco cells, protoplast preparation, and inoculation, and dot-blot assays for TMV-RNA were carried out essentially as described by Sela et al. (LZ), who recently reported a procedure for detecting TMV-RNA at the picogram level. Proto-
i Author to whom requests for reprints should be addressed. a Part of this work was carried out by the indicated authors at Roche Institute of Molecular Biology, Nutley, N. J. 07110.
173
0042-6822/85 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
SHORT
174
COMMUNICATIONS
plasts were prepared from cultured cell suspensions of tobacco var. Wisconsin 38. Determination of TMV coat protein in protoplasts was carried out by the enzymelinked immunosorbent assay (ELISA) as described by Orchansky et aa (6). Sedimented protoplasts were diluted 1:3 in a mannitol-free ELISA sample buffer and homogenized. A standard TMV dilution curve was included in every experiment. TMV-RNA accumulation in protoplasts was followed by removing aliquots at various time intervals after inoculation with TMV. Protoplasts were lysed, dotted on nitrocellulose, and hybridized with cDNA probes specific to TMV-RNA as described by Sela et al. (1.2). After autoradiography films were scanned in a densitometer (Cliniscan, Helena Laboratories) or alternatively, the dots were cut out and their radioactivity was measured in a liquid scintillation spectrometer.
Interferons
and interferon
treatments.
All interferon preparations were highly purified: (i) subspecies 73 (2.4 X lo8 units/ mg) of Rubinstein et al. (13) was obtained from M. Rubinstein, Weizmann Institute of Science, Rehovot, Israel. (ii) Recombinant human leukocyte interferon preparation IFN-aD (2.2 X lo8 units/mg), equivalent to the above-mentioned natural interferon 73, was obtained from S. Pestka, Roche Institute of Molecular Biology, Nutley, New Jersey. (iii) Recombinant interferon preparation IFN-aA (2.9 X lOa units/ mg), equivalent to the natural interferon cr2/31, was obtained from the Department of Molecular Genetics, Roche Research Center, Nutley, New Jersey. For information about classification and terminology of human leukocyte interferons see Pestka (1). The recombinant interferon preparations were obtained as described by Staehelin et al. (14) and Rehberg et
aL (15). Interferon was diluted to the desired concentration with the Murashige and Skoog medium as specified elsewhere (12) containing 12% mannitol and 1% fetal calf serum (FCS). Interferon at lo-fold the desired concentration (0.1 vol) was added to a 0.9 vol of protoplasts, so that the
final FCS concentration was always 0.1%. Higher concentrations of FCS caused the appearance of filamentous structures and protoplasts aggregation. A similar interferonless solution containing mannitol and FCS served as a mock interferon preparation. A polyclonal antibody preparation against interferon IFN-aA was obtained from S. Pestka. One milliliter of this preparation was sufficient to neutralize 50% of the activity of 1.7 X lo6 interferon units. In order to test the effect of these antibodies on interferon activity in tobacco protoplasts, 8500 antibody units were incubated with 1000 interferon units for 15 min at 37”. The interferon was then further diluted (103-106-fold) and applied to the protoplasts. Tobacco protoplasts were prepared, as described, from cell suspensions (Wisconsin 38) or leaf mesophyll (“Samsun”; “Samsun NN”). The Wisconsin 38 protoplasts were maintained at lo6 cells/ml and their virus content was determined by ELISA ‘72 hr after TMV inoculation. The mesophyll protoplasts were shaken as suspensions and their virus titers were determined by both ELISA and infectivity 48 hr postinoculation. Pretreatment with all three subspecies of leukocyte interferon, added to a final concentration of 1 unit/ml, protected three kinds of protoplasts from subsequent TMV infection (Table 1). Some protection was observed even after brief exposure to interferon (15-30 min), but the antiviral effect became substantial only after l-3 hr. When added 3 hr post-TMV inoculation, interferon had a much lesser effect, requiring a longer time in order to protect against viral infection (data not shown). In practice, interferon was found to confer maximum protection also when added within 1 hr after TMV inoculation (Fig. 2). In order to investigate the effect of interferon on TMV-RNA accumulation, protoplasts were pretreated with interferon IFN-aA (1 unit/ml, 3 hr) and then washed and inoculated with TMV. At various times postinoculation, aliquots were
-
-
381 (0)f
380 (0.3)
241 (38)
192 (51)
89 (77)
69 (82)
54 (86) 48 (88)
-
No additions
Mock, 12 hr
15 min
30 min
1 hr
3 hr
6 hr
12 hr
24 hr
TMV/lO”
Nanograms
-
of Nicotiana
30 (87)
-
38 (83) -
-
81 (79) 40 (90) 30 (92)
230 (0) 124 (46) -
245 (37) 176 (55)
-
-
glutinosa
and the number
30 (86)
-
31 (85)
211 (0) 110 (48) -
-
INFECTION
3 (92)
-
of 12 leaves).
2 (86)
-
5 (64) -
-
7 (81)
14 (0) 8 (43) -
-
Samsun NNd treated with y3
37 (0) 18 (51) -
-
Samsund treated with y3
Number of local lesions caused by protoplasts homogenatese
ON TMV
of lesions was counted (average
Samsun NNd treated with y3
protoplasts”
LEUKOCYTE INTERFERONS OF TOBACCO PROTOPLASTS
Samsund treated with y3
VARIANTS
IFN-aD
38’
1
WITH VARIOUS TYPES OF HUMAN IN SEVERAL
’ 1 unit/ml of the indicated type of interferon. *Determined by ELISA. ‘Prepared from cell suspensions. d Mesophyll protoplasts. e Protoplast homogenates (106/ml) were applied to half-leaves ‘Numbers in parentheses are percentage protection values.
30 (92) -
54 (86)
90 (77)
252 (34)
381 (19)
IFNCXA
Wisconsin
Treated with interferon Y3
Time of exposure to interferon” (prior to inoculation)
THE EFFECT OF PRETREATMENTS
TABLE
SHORT
176
COMMUNICATIONS
a Protoplasts (Wisconsin 38) were pretreated for 3 hr with 1 unit/ml of the interferon subspecies IFNolA, washed, and then inoculated with TMV.
lysed, and samples, equivalent to 25,000 cells per dot, were applied to nitrocellulose and hybridized with a cDNA probe as described. Interferon was found to be most active in preventing TMV-RNA replication in tobacco protoplasts at concentrations of 0.01-0.1 units/ml, inhibiting about 50% of TMV-RNA replication even at a concentration of 0.001 unit/ml. Increasing the interferon concentration above 1.0 unit/ml decreased its antiviral activity, but did not abolish it (Fig. 1). Experiments, similar to the above-described dose-response assays, were carried out, but the interferon was added immediately after rather than before TMV inoculation. In addition, part of the interferon preparation (1000 units/ml) was incubated with an anti-interferon antibody, at levels sufficient to neutralize four times the interferon concentration. The antibody-treated interferon was then diluted to 0.001-l units/ml and applied to the protoplasts. Protoplasts were then lysed,
taken for either dot-blot hybridization with a cDNA probe made from TMVRNA or TMV coat protein determination by ELISA. Following interferon treatment TMVRNA accumulation was almost totally inhibited for at least 60 hr after inoculation and was then resumed at a slow rate so that at 120 hr postinfection a 60% inhibition was still observed (Table 2). TMV coat antigen accumulation, initially inhibited, resumed at a faster rate, reaching control levels 120 hr after infection. It seems that for the coat protein expression to proceed normally the TMV-RNA within the cell has to reach only a certain threshold level. All further experiments were carried using the dot-blot hybridization assay system because of its greater sensitivity. Protoplast dose-response to interferon was studied as follows: Protoplasts were pretreated with various concentrations of interferon IFN-CYA for 3 hr, and then washed and inoculated with TMV. At ‘72 hr after inoculation protoplasts were
FIG. 1. The effect of interferon dosage on TMVRNA accumulation in tobacco protoplasts. Suspension cell protoplasts were exposed to interferon (recombinant IFN-aA) for 3 hr, inoculated with TMV, incubated for 72 hr, lysed, and their nucleic acids were denatured as described in the text. Aliquots, equivalent to 25,000 cells per dot were applied to nitrocellulose and hybridized with a cDNA probe specific to TMV-RNA. The autoradiogram, presented for comparison, was scanned in a densitometer.
TABLE
2
ACCIJMULATION OF TMV-RNA (DOT HYBRIDIZATION) AND OF TMV COAT PROTEIN ANTIGEN (ELISA) IN TOBAKO PROTOPLASTS FOLLOWING INTERFERON TREATMENT”
post-TMV inoculation (hr)
Time
30
60
SO
120
Dot hybridization (cpm/dot) No additions Mock interferon Interferon
62 42 7
279 242 87
467 440 172
634 726 236
ELISA (ng/106 cells) No additions Mock interferon Interferon
10 0 0
300 278 24
446 481 341
605 640 631
SHORT
177
COMMUNICATIONS
ab
ob
ab
l b
IllTERFEROlIr~il~/mll FIG. 2. The effect of anti-interferon antibodies on interferon activity in tobacco protoplasts. Interferon, either untreated or preincubated with anti-interferon antibodies, was applied to tobacco protoplasts 20 min after inoculation. Analyses are as in Fig. 1. (ab = antibodies).
treated, blotted, and hybridized as before. It was clearly demonstrated that the antibody treatment abolished the inhibitory effect of interferon and returned TMVRNA accumulation to the level of control infections (Fig. 2). The antibody preparation at the employed dilutions did not inhibit TMV increase (data not shown). This communication presents evidence that human leukocyte interferon suppresses TMV accumulation in tobacco protoplasts in addition to the previously reported inhibition in tobacco leaf disks (6). It is also demonstrated that a number of different interferon preparations, including recombinant species, are similarly active in protoplasts deriving from three different sources. In a protoplast system, cells which had been exposed to interferon exhibited resistance to subsequent virus infection, demonstrating that an antiviral state did indeed take place. The buildup of this antiviral state commenced almost instantaneously, but 3-6 hr of exposure to interferon were required for maximum effect. In this study TMV-RNA accumulation was measured directly, adding a new parameter to the hitherto assayed infectivity and coat protein antigen accumulation. The data presented in Table 2 suggest
that TMV-RNA replication is a primary target for the antiviral activity of interferon in tobacco. The dose-response of tobacco protoplasts to interferon was similar to that reported for tobacco leaf disks (6), except that in the protoplast system interferon concentrations above l-10 units/ml exhibited less antiviral activity. The protoplast system allows accurate determination of the interferon level required for inhibiting TMV-RNA replication. Assuming a sp act of 2 X lo* units of interferon per milligram protein and a molecular weight of 20,000, only one or a few molecules of interferon seem sufficient to confer maximum protection on a tobacco cell. The various interferon preparations tested were essentially pure proteins. Therefore it is highly improbable that there was an antivirally active contaminant in the diluted preparations. The neutralization of the antiviral activity of interferon by the corresponding antibody makes any such possibility unlikely and confirms the reliability of the assay. REFERENCES 1. PESTKA, S., Arch.
(1983). 2 SELA, I., Adv.
virus
B&hem
Biophgs.
Res. 26,201-237
221,
(1981).
1-37
178
SHORT
8 LOEBENSTEIN, 4. 5. 6. 7. 8.
9. 10.
G.,
and
GERA,
A.,
Virology
COMMUNICATIONS
114,
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