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The effects of oxidized phenolic compounds on the infectivity of four “stable” viruses
VIROLOGY
The
40, 540-546
Effects
(1970)
of Oxidized
Phenolic Four
Compounds
“Stable”
I<. N. SAKSENA Department
of Plant
Pathology, Washington and Extension Center, Accepted
on
the
Infectivity
of
Viruses’ AND
G.
I.
MINIi”
State University, Irrigated Prosser, Washington 99350 October
Agriculture
Research
24, 1969
Alfalfa mosaic virus (AMV), potato virus X (PVX), sowbane mosaic virus (SMV), and tobacco mosaic virus (TMV) were incubated for various intervals with commercial mushroom tyrosinase and 2 X 10-3 M catechol, dihydroxyphenylalanine (DOPA), or chlorogenic acid (CA) and tested for infectivity by direct assays and by assays of repurified virus. Alfalfa mosaic virus was totally inactivated in less than 10 min by oxidized catechol, but was not inactivated by oxidized DOPA or CA. Both PVX and SMV were partially inactivated by oxidized catechol, but were not inactivated by oxidized CA. SMV was partially inactivated by oxidized DOPA, but PVX was not. TMV was not inactivated by any of the three phenols tested. The level of inactivation of each virus exposed to oxidized catechol was reproduced when that virus was exposed to similar concentrations of tetrachloro-o-benzoquinone (TCQ). The results suggest that inactivation resulted from reactions between virus and the o-benzoquinone molecule rather than other possible oxidative intermediates. As the o-quinone molecule became more complex, its ability to inactivate a wide range of viruses decreased. Comparisons between assays made directly in the oxidized solutions and assays made after repurification suggest that oxidized phenols have little, if any, effect on host susceptibility when applied as part of the inoculum.
tivity to oxidized phenols. At least five viruses that lose infectivity rapidly in crude extracts have been shown to be irreversibly inactivated by oxidized phenolic compounds (Hampton and Fulton, 1961; Harrison and Pierpoint, 1963; Mink, 1965). Certain other unstable viruses are suspected of being sensitive to oxidized phenols because the addition of antioxidants or reducing agents to tissue extracts prevents an otherwise rapid loss of infectivity (Bald and Samuel, 1934; Best, 1939; Fulton, 1949; Fulton, 1952; Bawden and Pirie, 1957; Mink et al., 1966b). Because phenolic compounds occur widely in plants and because phenol oxidase activity in extracts of virus-infected tissues is often higher than in healthy tissues (Hampton and Fulton, 1961; Martin, 1958; van Hammen and Brouwer, 1964), it is often assumed that stable viruses are insensitive to oxi-
INTRODUCTION
Viruses are often referred to as “stable” based on their behavior in or “unstable” plant juice. The stable viruses generally retain most of their infectivity for days or even years in tissue extracts, whereas unstable viruses lose most, if not all, infectivity, within a few minutes or hours. Where studies have been made there appears to be a close correlation between rapid loss of infectivity in tissue extracts and virus sensi1 This investigation was supported in part by National Science -Foundation Grant GB-5639. Scientific paper No. 3330, Washington State University College of Agriculture, Pullman, Project No. 1887. 2 Research associate (current address: Department of Plant Sciences, University of Alberta, Edmonton, Alberta, Canada) and associate plant pathologist, respectively. 540
PHENOLIC
COMPOUNDS
EFFECT
dized phenols. However, there is little direct evidence to indicate whether or not this is universally true. Tobacco mosaic virus (TMV) (Bawden and Pirie, 1957), lucerne mosaic, tomato black ring, and carnation ringspot viruses (Harrison and Pierpoint, 1963) as well as southern bean mosaic virus (SBIMV) (Mink et al., 1963) have all been shown to be insensitive to oxidizing conditions in plant tissue that inactivated several unstable viruses. However, only SBMV was tested by exposing purified virus to a known oxidized phenol. In this case infectivity was not lost during oxidation even though the virus became highly colored (Mink et al., 1963). For this study we selected four viruses from among the many reported in the literature that retain infectivity for long periods in tissue homogenates. Alfalfa mosaic virus (AMV) and potato virus X (PVX) remain infectious for more than 7 and 234 days, respectively, in Nicotiana tabacum L. juice (Ross, 1941; Vasudeva and Lal, 1945). Sowbane mosaic virus (SMV) and tobacco mosaic virus (TMV) remain infectious in plant juice for more than 2 months and 50 years, respectively (Bennett and Costa, 1961; Silber and Burk, 1965). The sensitivity of each virus was tested to three enzymatically oxidized phenols-catechol, dihydroxyphenylalanine (DOPA), and chlorogenic acid. To avoid many of the uncertainties inherent in experiments with tissue extracts of unknown composition, experiments were made with purified viruses, commercial phenol oxidase (tyrosinase), and commercial phenolic compounds. MATERIALS
AND
METHODS
Preparation and assay of viruses. Alfalfa mosaic virus (ATCC-106; J. B. Bancroft, Lafayette, Indiana) and PVX (severe isolate; W. G. Hoyman, Prosser, Washington) were increased for purification in N. tabacum L. ‘Connecticut Havana 423’. Sowbane mosaic virus and TMV (local isolates) were increased in Chenopodium quinoa Willd. Infected leaf tissue was homogenized in a solution containing 0.01 M sodium diethydithiocarbamate (NaDIECA) and 0.01 M cysteine.HCI. Neither the antioxidant nor
ON
FOUR
“STABLE”
VIRUSES
541
the reducing agent was required to obtain highly infectious preparations of any of the 4 viruses. However, both compounds were included in all tissue homogenates to prevent or reduce the occurrence of undesirable oxidation reactions during extraction and clarification. Tobacco tissue homogenates containing A?(,‘IV were clarified by the chloroformbutanol procedure (Steere, 1956). Homogenates containing PVX were clarified with activated charcoal and centrifugation (Galvez, 1964). Homogenates of C. quinoa tissue containing either SMV or TMV were clarified by heat (65°C for 15 min) and low speed centrifugation. Clarified preparations were given 2 cycles of differential centrifugntion and the pellets were suspended in 1O-2 M neutral phosphate buffer. Each preparation was then sedimented 2 hours at 24,000 rpm on rate sucrose density-gradient columns (Brakke, 1953) in an SW 25.1 rotor and a Spinco Model L preparative centrifuge. For each virus only the infectious zone corresponding to whole virus was removed from density-gradient tubes and concentrated in 1O-2 ill neutral phosphate buffer by differential centrifugation. Virus concentrations were estimated from optical densities at 260 rnp using extinction values of 5.0, 2.9, 4.0, and 3.2 for 1 mg/ml with a l-cm light path for AMV, I’VX, SMV, and TRIV, respectively. The concentration of purified virus uesd in each series of experiments was selected on the basis of dilution assays. Solutions were adjusted so that final dilutions of control treatments produced between 50 and 150 lesions per half-leaf. Infectivity assays for AR/IV, PVX, SMV, and TMV were made on preshaded, Carborundum-dusted leaves of Phaseolus vulgaris L. ‘Bountiful’, C. quinoa, C. amaranticolor Coste & Reyn., and N. tabacum ‘423’, respectively. Preparation of enzyme and substrate. Commercial mushroom tyrosinase (Nutritional Biochemical Corporation) was dissolved in distilled water (0.2 mg/ml) and refrigerated until use within 2448 hours. Enzymatic activity against each phenolic substrate was determined before each experiment at 24°C. Two milliliters of the appropriate poly-
542
SAKSENA
phenol (2.5 X 1OW M in 1OW M neutral phosphate buffer) were placed in a l-cm silica cell in a Beckman DB-G spectrophotometer preset at the E,,, for the respective o-quinones (390 rnp for catechol and CA, 470 rnp for DOPA). The reference cell contained 2 ml of phosphate buffer. Stock enzyme solution (0.5 ml) was added to each cell and mixed thoroughly; the recorder was started simultaneously with enzyme addition to the phenol. Catechol, DOPA, and chlorogenic acid were used without further purification and were dissolved in 10e2 M neutral phosphate buffer immediately before use.
AND
MINK TABLE
1
THE PERCENT DECREASE IN LESION NUMBERS OBTAINED WHEN MI~TIJRES OF VIRUS, TYROSINASE, AND PHENOL WERE ASSAYED DIRECTLY .IFTER 10 MIN OXIDATION Phenol Virus Catechol BMV PVX SMV TMV
2 x
DOPA
100 85 96 0
Chlorogenic acid
0 0 48 0
Q Phenol concentration 10-s M.
during
0 0 0 0 incubation
was
RESULTS
E$ect of Polyphenol Oxidation m Infectivity Preliminary tests were made to determine the sensitivity of AMV, PVX, SMV, and TMV to polyphenol oxidation using essentially the direct assay technique described earlier (Mink, 1965). One milliliter of solutions containing 60 pg/ml AMV, 35 pg/ml PVX, 475 pg/ml SMV, or 150 kg/ml TMV in lo-” M neutral phosphate buffer were mixed with an equal volume of the appropriate phenol at 5 X low3 M in phosphate buffer. Stock enzyme solution (0.5 ml) was added and mixed thoroughly. Immediately after enzyme addition (usually within 5 set) and at preselected time intervals 0.6 ml of the mixture was transferred to 0.2 ml of 0.01 d1 NaDIECA to stop enzymatic activity. In early experiments we attempted to reduce oxidized compounds present with sodium dithionite (Mink, 1967) before assays were made. However, the reducing agent inactivated some viruses, particularly PVX. In addition, different phenols when oxidized produced different intermediates, some of which could not be reduced after the reactions had progressed several minutes. Consequently, for preliminary tests all treatments were assayed directly in the unreduced reaction mixture on the appropriate hosts. In only 4 of the 12 virus-phenol combinations were there reduced lesion numbers when assays were made after 10 min oxidation (Table 1). Only TMV solutions were unaffected by any of the 3 polyphenols
TABLE
2
EFFECT OF THE ASSAY PROCEDURE ON THE PERCENT DECREASE IN LESION NUMBERS OBTAINED AFTER 10 MIN OXIDATION Assay Virus
AMV PVX SMV SMV
procedure
Phenol
Catechol Catechol Catechol DOPA
Direct
Purified
100 85 90 55
100 80 91 36
tested. Oxidized catechol reduced lesion numbers of AMV, PVX, and SMV solutions, whereas oxidized DOPA reduced lesions only of SMV. Chlorogenic acid oxidation had no effect on solutions of any of the 4 viruses tested. To determine whether the decreased lesion numbers obtained in the 4 “active” virusphenol combinations was due to direct effects of oxidative products on the viruses or to effects of oxidative intermediates on assay host susceptibility, experiments were performed as above using 3-10 times the volumes indicated. After enzymatic activity was stopped, a sample was removed for direct assay and the remainder of each treatment was centrifuged at 39,000 rpm for 1.5 hours. Virus pellets were suspended in 0.01 M neutral phosphate buffer and assayed. Similar results were obtained by either assay procedure (Table 2). Furthermore, when samples of the oxidizing mixtures were removed and tested at pre-
PHENOLIC
COMPOUNDS
EFFECT
80
5 560 2 5 $40
ae 20
MINUTES FIG. 1. The effect of the assay procedure on the measured rate of AMV inactivation in the presence of tyrosinase oxidized catechol. l = direct assay, 0 = virus repurified before assay.
ON
FOUR
“STABLE”
VIRUSES
543
pH. Figure 2 illustrates the spectrophotometric sequence of events that occurred under the conditions used for our inactivation assays. At catechol concentrations between 3 X lop4 M and 4 X lop4 M more than 90% of the polyphenol was oxidized to o-benzoquinone within 60 set (Fig. 3). However, at catechol concentrations near 2 X 1O-3 2M only l&25% (depending upon the activity of the enzyme preparation) of the catechol was oxidized within the first minute. In addition, approximately 35% of the o-benzoquinone formed was rapidly reduced back to nonoxidized forms (Fig. 3). As a result, approximately the same concentration of o-benzoquinone appeared to be present after 1 min in solutions initially containing either 4 X 10e4 M or 2 X 1OWiW catechol. Regardless of the initial concentration used, inactivation of AMV in the presence
selected intervals by both assay procedures, the percentage decrease in lesion numbers was essentially the same. Fig. 1 illustrates the results obtained when catechol was oxidized in the presence of AMV. Each virus-phenol combination was tested and comparable results obtained. The results of these studies indicate that AMV, PVX, and SMV are each inactivated in the presence of catechol oxidation and that SMV is also partially inactivated in the presence of DOPA oxidation. In addition, the results indicate that the compounds formed during the first 10 min of catechol, DOPA, and chlorogenic acid oxidation do not affect susceptibility of the 4 assay hosts when applied to the leaf surface simultaneously with the virus. Kinetics of Polyphenol OxicZatim and Virus Inactivatim The initial step in tyrosinase-oxidation of catechol is the formation o-benzoquinone which, in aqueous solutions, polymerizes to form an ill-defined series of intermediates ending with catechol melanin (Mason, 1949). During the early stage of oxidation the amount of o-benzoquinone that is produced in excess of that consumed in subsequent reactions is dependent not only upon the concentrations of reactants, but also upon
240 FIG. 2. The spectrophotometric course of events during tyrosinase oxidation of 3.16 X 10v4 jW catechol. Broken line = before oxidation; solid lines 1, 8, S, and 4 = immediately, 3, 6, and 9 min, respectively, after enzyme addition.
544
SAKSENA
AND
# -20 / 4 1
2
3
4
5
MI NUTES FIG. 3. Tyrosinase oxidation (line 1) and 2 X lO+ M (line 2) rate of AMV inactivation during 3.3 X 1W4 M (0) and 2 X 1OF M
of 3.3 X 1OW M catechol and the the oxidation of (0) catechol.
MINK
2,3-dihydroindate-5,6 quinone) is formed which is relatively stable at neutral pH but which eventually rearranges to form 5,6-dihydroxyindole and finally DOPA melanin (Jlason, 1948). Under our conditions once oxidation had occurred and the spectrum corresponding to the red compound had been obtained, we found that no other spectrophometric changes occurred for at least 30 min at room temperature. Inactivation of SMV did not parallel the measured increase in DOPA oxidation (Fig. 5). The apparent nonassociation between rates of virus inactivation and quinone formation suggests that inactivation did not occur during the ‘enzymatic transfer of electrons, but rather occurred as a result of chemical reactions between virus and one or more oxidation products. Virus Inactivation quinone
by
Tetrachloro-o-benxo-
Spectrophotometric data suggest o-benzoquinone was the main oxidized compound present during the first 10 min of catechol oxidation. In nearly all tests the amount of o-benzoquinone produced within l-2 min was calculated to be between approximately 30 and 35 pg/ml. However, each virus was tested at a different concentration. Consequently, the ratio of quinone to virus varied 123456189lf) MINUTES 4. Inactivation SMV (0) in the
FIG.
and tion.
of AMV (a), PVS (*), presence of catechol oxida-
of oxidizing catechol did not parallel the formation of o-benzoquinone measured spectrophotometrically. o-Benzoquinone formation was essentially complete in approximately 60 set while complete inactivation of AMV required between 5 and 10 min (Fig. 3). Similar results were obtained with SMV and PVX. However, these two viruses were only partially inactivated within 5 min and retained small amounts of infectivity after 10 min oxidation (Fig. 4). When DOPA is oxidized in the presence of tyrosinaae, a red compound (2-carboxy-
a E
.9 .
_,_,.___._....-.---.. /.. . . . .
2
190 -80
20
5
MINhES FIG. 5. Tyrosinase (broken line) DOPA (solid line).
oxidation of 5 X lo+ M and inactivation of SMV
PHENOLIC
COMPOUNDS
TABLE IN.~CTIVATION PRODUCED
3
OF 4 VIRUSES 0-BENZOQUINONE
BY
ENZYMATICSLLY
AND
580 1017 77 200
100 85 90 0
CHEMICALLY
(TCQ)
PREP.~REDTETR.XHLORO-0-BENZOQUINONE
AMV PVX SMV TMV
EFFECT
500 1000 80 200
10 80 50 0
among viruses. The calculated ratios were 550,1017,77, and 210 pg o-benzoquinone/mg of AMV, PVX, SMV, and TMV, respectively. To determine whether or not the measured amounts of o-benzoquinone were sufficient to produce the levels of inactivation obtained with each virus, tests were made with tetrachloro-o-benzoquinone (TCQ) by methods described earlier (Mink, 1967) at quinone to virus ratios comparable to those calculated above. The results indicate that the amounts of o-benzoquinone formed during catechol oxidation were sufficient to produce the levels of inactivation measured with all 4 viruses (Table 3). DISCUSSION
A wide variety of phenolic compounds have been found in plant tissues (Harborne, 1964). These compounds vary not only in their suitability as substrate for phenol oxidases, but also in their ability to react with different chemical groups when oxidized (Mason, 1955). Variation in the structure of o-quinones was previously demonstrated (Mink et al., 1966a) to have a pronounced effect on their ability to inactivate Tulare apple mosaic virus (TAMV). Consequently to consider virus sensitivity or insensitivity to “oxidized phenols” as if all oxidized phenols behaved similarly appears to oversimplify a somewhat complex situation. Sensitivity to oxidize phenols appears to depend not only upon the virus but also upon the phenol tested. Earlier studies indicated that cucumber mosaic virus (Harrison and Pierpoint, 1963) and TAMV (Mink, 1965) were readily inactivated by enzymatically oxidized chlorogenic acid. How-
ON
FOUR
“STABLE”
VIRUSES
545
ever, this phenol had no effect when oxidized on the infectivity of the 4 viruses tested here, despite the fact that 3 of the 4 viruses were readily inactivated by oxidized catechol. Furthermore, of the 4 viruses we tested, only SllIV was inactivated by oxidized DOPA, even though this compound was used earlier to inactivate 3 stone fruit viruses (Hampton and Fulton, 1961) and TAMV (Mink, unpublished). It is apparent that even though rapid loss of infectivity in tissue extracts may indicate viruses that are sensitive to an oxidized phenol, the reverse is not necessarily true. The stability of an otherwise unstable virus may result from the scarcity of an “active” phenol. For example, TAMV is highly senitive to oxidized chlorogenic acid and loses infectivity rapidly in tobacco leaf extracts where the primary phenol is chlorogenic acid (Zucker and Ahrens, 1958). Both AMV and PVX are insensitive to oxidized chlorogenie acid and are stable in tobacco juice. Except possibly for the report of Harrison and Pierpoint (1963)) all viruses that have been demonstrated to be inactivated by oxidized phenolic compounds appear to be inactivated by the o-benzoquinone structure rather than some transient intermediate This is substantiated by the fact that TCQ, which presumably participates only in oxidation-reduction reactions, can be used to produce predictable levels of inactivation. The results obtained here indicate that as the o-quinone structure becomes more complex (i.e., catechol, DOPA, anp chlorogenic acid), it becomes limited in the number of viruses it can inactivate. In previous tests when the molecular size of 4methyl-o-benzoquinone was increased by attachment to ovalbumin protein, that compound lost its ability to inactivate TAMV (Mink et al., 1966a). The available data continue to suggest that the steric configuration of the quinone and perhaps the structural integrity of virus proteins determine virus susceptibility to a particular o-quinone. However, it has not yet been established whether inactivation results from reactions between o-quinones and virus ribonucleic acid (RNA) or from a “tanning” of the virus protein. If RNA alone is in-
546
SAKSENA
volved it would appear that the permeability of virus proteins to low molecular weight compounds, such as quinones, varies considerably among viruses. REFERENCES J. G., and SAMUEL, G. (1934). Some factors affecting the inactivation rate of the virus of tomato spotted wilt. Ann. Appl. Biol. 21, 179190. BAWDEN, F. C., and PIRIE, N. W. (1957). A virus inactivating system from tobacco leaves. J. Gen. Microbial. 16,697-710. BENNETT, C. W., and COSTA, A. S. (1961). Sowbane mosaic caused by a seed-transmitted virus. Phytopathology 51,546-550. BEST, R. J. (1939). The preservative effect of some reducing systems on tomato spotted wilt virus. Sustralian J. Exptl. Biol. Med. Sci. 17, l-7. BRAKKE, M. K. (1953). Zonal separations by density-gradient centrifugation. Arch. Biochem. Biophys. 45, 275-290. FULTON, R. W. (1649). Virus concentration in plants acquiring tolerance to tobacco streak. Phytopathology 39, 231-243. FULTON, R. W. (1952). Mechanical transmission and properties of rose mosaic virus. Phytopathology 42, 413316. G~LVEZ, G. E. (1964). Loss of virus by filtration through charcoal. Virology 23, 307312. HAMPTON, R. E., and FULTON, R. W. (1961). The relation of polyphenol oxidase to instability in vitro of prune dwarf and sour cherry necrotic ringspot viruses. Virology 13, 4452. HARBORNE, J. B., ed. (1964). “Biochemistry of Phenolic Compounds.” Academic Press, New York. HARRISON, B. D., and PIERPOINT, W. S. (1963). The relation of polyphenoloxidase in leaf extracts to the instability of cucumber mosaic and other plant viruses. J. Gen. Microbial. 32, 417427. MARTIN, C. M. (1958). &ude cornparke de 1 activitb de la polyphdnoloxidase chew le tabac sain et inocule par une maladie B virus. Compt. Rend. Acad. Sci. 246, 2026-2029. BALD,
AND
MINK
H. S. (1948). The chemistry of melanin. III. Mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. J. Biol. Chem. 172, 83-99. MASON, H. S. Q949). The chemistry of melanin. IV. Mechanism of the oxidat,ion of catechol by tyrosinase. J. Biol. Chem. 181,803~812. MASON, H. S. (1955). Comparative biochemistry of the phenolase complex. Advan. Enzymol. 16, 105-184. MINK, G. I. (1965). Inactivation of Tulare apple mosaic virus by o-quinones. Virology 26,700-707. MINK, G. I. (1967). Kinetics of quinone inactivation of Tulare apple mosaic virus. Virology 33, 609-612. MINK, G. I., BANCROFT, J. B., and NADAK.IVUKAREN, M. J. (1963). Properties of Tulare apple mosaic virus. Phytopathology 53, 973-978. MINK, G. I., HUISMAN, O., and SAKSENA, K. N. (1966a). Oxidative inactivation of Tulare apple mosaic virus. Virology 29, 437-443. MINK, G. I., SAKSENA, K. N., and SILBERNAGEL, M. J. (1966b). Purification of the bean red node strain of tobacco streak virus. Phytopathology 56, 645-649. Ross, A. F. (1941). Purification and properties of alfalfa-mosaic virus protein. Phytopathology 31, 394-410. SILBER, G., and BURK, L. G. (1965). Infectivity of tobacco mosaic virus stored for 50 years in extracted “unpreserved” plant juice. Nature 206, 740-741. STEERE, R. L. (1956). Purification and properties of tobacco ringspot virus. Phytopathology 46, 60-69. VAN KA~MEN, A., and BROUWER, D. (1964). Increase of polyphenol oxidase activity by a local virus infection in uninoculated parts of plants. Virology 22, 9-14. VASUDEVA, R. S., and L~L, T. B. (1945). Studies on the virus diseases of potatoes in India. I. Occurrence of Solanum virus 1. Indian J. ,4gr. Sci. 14, 28&295. ZUCKER, M., and AHRENS, J. F. (1958). Quantitative assay of chlorogenic acid and its pattern of distribution within tobacco leaves. Plant Physiol. 33, 246-249. MASON,
The
40, 540-546
Effects
(1970)
of Oxidized
Phenolic Four
Compounds
“Stable”
I<. N. SAKSENA Department
of Plant
Pathology, Washington and Extension Center, Accepted
on
the
Infectivity
of
Viruses’ AND
G.
I.
MINIi”
State University, Irrigated Prosser, Washington 99350 October
Agriculture
Research
24, 1969
Alfalfa mosaic virus (AMV), potato virus X (PVX), sowbane mosaic virus (SMV), and tobacco mosaic virus (TMV) were incubated for various intervals with commercial mushroom tyrosinase and 2 X 10-3 M catechol, dihydroxyphenylalanine (DOPA), or chlorogenic acid (CA) and tested for infectivity by direct assays and by assays of repurified virus. Alfalfa mosaic virus was totally inactivated in less than 10 min by oxidized catechol, but was not inactivated by oxidized DOPA or CA. Both PVX and SMV were partially inactivated by oxidized catechol, but were not inactivated by oxidized CA. SMV was partially inactivated by oxidized DOPA, but PVX was not. TMV was not inactivated by any of the three phenols tested. The level of inactivation of each virus exposed to oxidized catechol was reproduced when that virus was exposed to similar concentrations of tetrachloro-o-benzoquinone (TCQ). The results suggest that inactivation resulted from reactions between virus and the o-benzoquinone molecule rather than other possible oxidative intermediates. As the o-quinone molecule became more complex, its ability to inactivate a wide range of viruses decreased. Comparisons between assays made directly in the oxidized solutions and assays made after repurification suggest that oxidized phenols have little, if any, effect on host susceptibility when applied as part of the inoculum.
tivity to oxidized phenols. At least five viruses that lose infectivity rapidly in crude extracts have been shown to be irreversibly inactivated by oxidized phenolic compounds (Hampton and Fulton, 1961; Harrison and Pierpoint, 1963; Mink, 1965). Certain other unstable viruses are suspected of being sensitive to oxidized phenols because the addition of antioxidants or reducing agents to tissue extracts prevents an otherwise rapid loss of infectivity (Bald and Samuel, 1934; Best, 1939; Fulton, 1949; Fulton, 1952; Bawden and Pirie, 1957; Mink et al., 1966b). Because phenolic compounds occur widely in plants and because phenol oxidase activity in extracts of virus-infected tissues is often higher than in healthy tissues (Hampton and Fulton, 1961; Martin, 1958; van Hammen and Brouwer, 1964), it is often assumed that stable viruses are insensitive to oxi-
INTRODUCTION
Viruses are often referred to as “stable” based on their behavior in or “unstable” plant juice. The stable viruses generally retain most of their infectivity for days or even years in tissue extracts, whereas unstable viruses lose most, if not all, infectivity, within a few minutes or hours. Where studies have been made there appears to be a close correlation between rapid loss of infectivity in tissue extracts and virus sensi1 This investigation was supported in part by National Science -Foundation Grant GB-5639. Scientific paper No. 3330, Washington State University College of Agriculture, Pullman, Project No. 1887. 2 Research associate (current address: Department of Plant Sciences, University of Alberta, Edmonton, Alberta, Canada) and associate plant pathologist, respectively. 540
PHENOLIC
COMPOUNDS
EFFECT
dized phenols. However, there is little direct evidence to indicate whether or not this is universally true. Tobacco mosaic virus (TMV) (Bawden and Pirie, 1957), lucerne mosaic, tomato black ring, and carnation ringspot viruses (Harrison and Pierpoint, 1963) as well as southern bean mosaic virus (SBIMV) (Mink et al., 1963) have all been shown to be insensitive to oxidizing conditions in plant tissue that inactivated several unstable viruses. However, only SBMV was tested by exposing purified virus to a known oxidized phenol. In this case infectivity was not lost during oxidation even though the virus became highly colored (Mink et al., 1963). For this study we selected four viruses from among the many reported in the literature that retain infectivity for long periods in tissue homogenates. Alfalfa mosaic virus (AMV) and potato virus X (PVX) remain infectious for more than 7 and 234 days, respectively, in Nicotiana tabacum L. juice (Ross, 1941; Vasudeva and Lal, 1945). Sowbane mosaic virus (SMV) and tobacco mosaic virus (TMV) remain infectious in plant juice for more than 2 months and 50 years, respectively (Bennett and Costa, 1961; Silber and Burk, 1965). The sensitivity of each virus was tested to three enzymatically oxidized phenols-catechol, dihydroxyphenylalanine (DOPA), and chlorogenic acid. To avoid many of the uncertainties inherent in experiments with tissue extracts of unknown composition, experiments were made with purified viruses, commercial phenol oxidase (tyrosinase), and commercial phenolic compounds. MATERIALS
AND
METHODS
Preparation and assay of viruses. Alfalfa mosaic virus (ATCC-106; J. B. Bancroft, Lafayette, Indiana) and PVX (severe isolate; W. G. Hoyman, Prosser, Washington) were increased for purification in N. tabacum L. ‘Connecticut Havana 423’. Sowbane mosaic virus and TMV (local isolates) were increased in Chenopodium quinoa Willd. Infected leaf tissue was homogenized in a solution containing 0.01 M sodium diethydithiocarbamate (NaDIECA) and 0.01 M cysteine.HCI. Neither the antioxidant nor
ON
FOUR
“STABLE”
VIRUSES
541
the reducing agent was required to obtain highly infectious preparations of any of the 4 viruses. However, both compounds were included in all tissue homogenates to prevent or reduce the occurrence of undesirable oxidation reactions during extraction and clarification. Tobacco tissue homogenates containing A?(,‘IV were clarified by the chloroformbutanol procedure (Steere, 1956). Homogenates containing PVX were clarified with activated charcoal and centrifugation (Galvez, 1964). Homogenates of C. quinoa tissue containing either SMV or TMV were clarified by heat (65°C for 15 min) and low speed centrifugation. Clarified preparations were given 2 cycles of differential centrifugntion and the pellets were suspended in 1O-2 M neutral phosphate buffer. Each preparation was then sedimented 2 hours at 24,000 rpm on rate sucrose density-gradient columns (Brakke, 1953) in an SW 25.1 rotor and a Spinco Model L preparative centrifuge. For each virus only the infectious zone corresponding to whole virus was removed from density-gradient tubes and concentrated in 1O-2 ill neutral phosphate buffer by differential centrifugation. Virus concentrations were estimated from optical densities at 260 rnp using extinction values of 5.0, 2.9, 4.0, and 3.2 for 1 mg/ml with a l-cm light path for AMV, I’VX, SMV, and TRIV, respectively. The concentration of purified virus uesd in each series of experiments was selected on the basis of dilution assays. Solutions were adjusted so that final dilutions of control treatments produced between 50 and 150 lesions per half-leaf. Infectivity assays for AR/IV, PVX, SMV, and TMV were made on preshaded, Carborundum-dusted leaves of Phaseolus vulgaris L. ‘Bountiful’, C. quinoa, C. amaranticolor Coste & Reyn., and N. tabacum ‘423’, respectively. Preparation of enzyme and substrate. Commercial mushroom tyrosinase (Nutritional Biochemical Corporation) was dissolved in distilled water (0.2 mg/ml) and refrigerated until use within 2448 hours. Enzymatic activity against each phenolic substrate was determined before each experiment at 24°C. Two milliliters of the appropriate poly-
542
SAKSENA
phenol (2.5 X 1OW M in 1OW M neutral phosphate buffer) were placed in a l-cm silica cell in a Beckman DB-G spectrophotometer preset at the E,,, for the respective o-quinones (390 rnp for catechol and CA, 470 rnp for DOPA). The reference cell contained 2 ml of phosphate buffer. Stock enzyme solution (0.5 ml) was added to each cell and mixed thoroughly; the recorder was started simultaneously with enzyme addition to the phenol. Catechol, DOPA, and chlorogenic acid were used without further purification and were dissolved in 10e2 M neutral phosphate buffer immediately before use.
AND
MINK TABLE
1
THE PERCENT DECREASE IN LESION NUMBERS OBTAINED WHEN MI~TIJRES OF VIRUS, TYROSINASE, AND PHENOL WERE ASSAYED DIRECTLY .IFTER 10 MIN OXIDATION Phenol Virus Catechol BMV PVX SMV TMV
2 x
DOPA
100 85 96 0
Chlorogenic acid
0 0 48 0
Q Phenol concentration 10-s M.
during
0 0 0 0 incubation
was
RESULTS
E$ect of Polyphenol Oxidation m Infectivity Preliminary tests were made to determine the sensitivity of AMV, PVX, SMV, and TMV to polyphenol oxidation using essentially the direct assay technique described earlier (Mink, 1965). One milliliter of solutions containing 60 pg/ml AMV, 35 pg/ml PVX, 475 pg/ml SMV, or 150 kg/ml TMV in lo-” M neutral phosphate buffer were mixed with an equal volume of the appropriate phenol at 5 X low3 M in phosphate buffer. Stock enzyme solution (0.5 ml) was added and mixed thoroughly. Immediately after enzyme addition (usually within 5 set) and at preselected time intervals 0.6 ml of the mixture was transferred to 0.2 ml of 0.01 d1 NaDIECA to stop enzymatic activity. In early experiments we attempted to reduce oxidized compounds present with sodium dithionite (Mink, 1967) before assays were made. However, the reducing agent inactivated some viruses, particularly PVX. In addition, different phenols when oxidized produced different intermediates, some of which could not be reduced after the reactions had progressed several minutes. Consequently, for preliminary tests all treatments were assayed directly in the unreduced reaction mixture on the appropriate hosts. In only 4 of the 12 virus-phenol combinations were there reduced lesion numbers when assays were made after 10 min oxidation (Table 1). Only TMV solutions were unaffected by any of the 3 polyphenols
TABLE
2
EFFECT OF THE ASSAY PROCEDURE ON THE PERCENT DECREASE IN LESION NUMBERS OBTAINED AFTER 10 MIN OXIDATION Assay Virus
AMV PVX SMV SMV
procedure
Phenol
Catechol Catechol Catechol DOPA
Direct
Purified
100 85 90 55
100 80 91 36
tested. Oxidized catechol reduced lesion numbers of AMV, PVX, and SMV solutions, whereas oxidized DOPA reduced lesions only of SMV. Chlorogenic acid oxidation had no effect on solutions of any of the 4 viruses tested. To determine whether the decreased lesion numbers obtained in the 4 “active” virusphenol combinations was due to direct effects of oxidative products on the viruses or to effects of oxidative intermediates on assay host susceptibility, experiments were performed as above using 3-10 times the volumes indicated. After enzymatic activity was stopped, a sample was removed for direct assay and the remainder of each treatment was centrifuged at 39,000 rpm for 1.5 hours. Virus pellets were suspended in 0.01 M neutral phosphate buffer and assayed. Similar results were obtained by either assay procedure (Table 2). Furthermore, when samples of the oxidizing mixtures were removed and tested at pre-
PHENOLIC
COMPOUNDS
EFFECT
80
5 560 2 5 $40
ae 20
MINUTES FIG. 1. The effect of the assay procedure on the measured rate of AMV inactivation in the presence of tyrosinase oxidized catechol. l = direct assay, 0 = virus repurified before assay.
ON
FOUR
“STABLE”
VIRUSES
543
pH. Figure 2 illustrates the spectrophotometric sequence of events that occurred under the conditions used for our inactivation assays. At catechol concentrations between 3 X lop4 M and 4 X lop4 M more than 90% of the polyphenol was oxidized to o-benzoquinone within 60 set (Fig. 3). However, at catechol concentrations near 2 X 1O-3 2M only l&25% (depending upon the activity of the enzyme preparation) of the catechol was oxidized within the first minute. In addition, approximately 35% of the o-benzoquinone formed was rapidly reduced back to nonoxidized forms (Fig. 3). As a result, approximately the same concentration of o-benzoquinone appeared to be present after 1 min in solutions initially containing either 4 X 10e4 M or 2 X 1OWiW catechol. Regardless of the initial concentration used, inactivation of AMV in the presence
selected intervals by both assay procedures, the percentage decrease in lesion numbers was essentially the same. Fig. 1 illustrates the results obtained when catechol was oxidized in the presence of AMV. Each virus-phenol combination was tested and comparable results obtained. The results of these studies indicate that AMV, PVX, and SMV are each inactivated in the presence of catechol oxidation and that SMV is also partially inactivated in the presence of DOPA oxidation. In addition, the results indicate that the compounds formed during the first 10 min of catechol, DOPA, and chlorogenic acid oxidation do not affect susceptibility of the 4 assay hosts when applied to the leaf surface simultaneously with the virus. Kinetics of Polyphenol OxicZatim and Virus Inactivatim The initial step in tyrosinase-oxidation of catechol is the formation o-benzoquinone which, in aqueous solutions, polymerizes to form an ill-defined series of intermediates ending with catechol melanin (Mason, 1949). During the early stage of oxidation the amount of o-benzoquinone that is produced in excess of that consumed in subsequent reactions is dependent not only upon the concentrations of reactants, but also upon
240 FIG. 2. The spectrophotometric course of events during tyrosinase oxidation of 3.16 X 10v4 jW catechol. Broken line = before oxidation; solid lines 1, 8, S, and 4 = immediately, 3, 6, and 9 min, respectively, after enzyme addition.
544
SAKSENA
AND
# -20 / 4 1
2
3
4
5
MI NUTES FIG. 3. Tyrosinase oxidation (line 1) and 2 X lO+ M (line 2) rate of AMV inactivation during 3.3 X 1W4 M (0) and 2 X 1OF M
of 3.3 X 1OW M catechol and the the oxidation of (0) catechol.
MINK
2,3-dihydroindate-5,6 quinone) is formed which is relatively stable at neutral pH but which eventually rearranges to form 5,6-dihydroxyindole and finally DOPA melanin (Jlason, 1948). Under our conditions once oxidation had occurred and the spectrum corresponding to the red compound had been obtained, we found that no other spectrophometric changes occurred for at least 30 min at room temperature. Inactivation of SMV did not parallel the measured increase in DOPA oxidation (Fig. 5). The apparent nonassociation between rates of virus inactivation and quinone formation suggests that inactivation did not occur during the ‘enzymatic transfer of electrons, but rather occurred as a result of chemical reactions between virus and one or more oxidation products. Virus Inactivation quinone
by
Tetrachloro-o-benxo-
Spectrophotometric data suggest o-benzoquinone was the main oxidized compound present during the first 10 min of catechol oxidation. In nearly all tests the amount of o-benzoquinone produced within l-2 min was calculated to be between approximately 30 and 35 pg/ml. However, each virus was tested at a different concentration. Consequently, the ratio of quinone to virus varied 123456189lf) MINUTES 4. Inactivation SMV (0) in the
FIG.
and tion.
of AMV (a), PVS (*), presence of catechol oxida-
of oxidizing catechol did not parallel the formation of o-benzoquinone measured spectrophotometrically. o-Benzoquinone formation was essentially complete in approximately 60 set while complete inactivation of AMV required between 5 and 10 min (Fig. 3). Similar results were obtained with SMV and PVX. However, these two viruses were only partially inactivated within 5 min and retained small amounts of infectivity after 10 min oxidation (Fig. 4). When DOPA is oxidized in the presence of tyrosinaae, a red compound (2-carboxy-
a E
.9 .
_,_,.___._....-.---.. /.. . . . .
2
190 -80
20
5
MINhES FIG. 5. Tyrosinase (broken line) DOPA (solid line).
oxidation of 5 X lo+ M and inactivation of SMV
PHENOLIC
COMPOUNDS
TABLE IN.~CTIVATION PRODUCED
3
OF 4 VIRUSES 0-BENZOQUINONE
BY
ENZYMATICSLLY
AND
580 1017 77 200
100 85 90 0
CHEMICALLY
(TCQ)
PREP.~REDTETR.XHLORO-0-BENZOQUINONE
AMV PVX SMV TMV
EFFECT
500 1000 80 200
10 80 50 0
among viruses. The calculated ratios were 550,1017,77, and 210 pg o-benzoquinone/mg of AMV, PVX, SMV, and TMV, respectively. To determine whether or not the measured amounts of o-benzoquinone were sufficient to produce the levels of inactivation obtained with each virus, tests were made with tetrachloro-o-benzoquinone (TCQ) by methods described earlier (Mink, 1967) at quinone to virus ratios comparable to those calculated above. The results indicate that the amounts of o-benzoquinone formed during catechol oxidation were sufficient to produce the levels of inactivation measured with all 4 viruses (Table 3). DISCUSSION
A wide variety of phenolic compounds have been found in plant tissues (Harborne, 1964). These compounds vary not only in their suitability as substrate for phenol oxidases, but also in their ability to react with different chemical groups when oxidized (Mason, 1955). Variation in the structure of o-quinones was previously demonstrated (Mink et al., 1966a) to have a pronounced effect on their ability to inactivate Tulare apple mosaic virus (TAMV). Consequently to consider virus sensitivity or insensitivity to “oxidized phenols” as if all oxidized phenols behaved similarly appears to oversimplify a somewhat complex situation. Sensitivity to oxidize phenols appears to depend not only upon the virus but also upon the phenol tested. Earlier studies indicated that cucumber mosaic virus (Harrison and Pierpoint, 1963) and TAMV (Mink, 1965) were readily inactivated by enzymatically oxidized chlorogenic acid. How-
ON
FOUR
“STABLE”
VIRUSES
545
ever, this phenol had no effect when oxidized on the infectivity of the 4 viruses tested here, despite the fact that 3 of the 4 viruses were readily inactivated by oxidized catechol. Furthermore, of the 4 viruses we tested, only SllIV was inactivated by oxidized DOPA, even though this compound was used earlier to inactivate 3 stone fruit viruses (Hampton and Fulton, 1961) and TAMV (Mink, unpublished). It is apparent that even though rapid loss of infectivity in tissue extracts may indicate viruses that are sensitive to an oxidized phenol, the reverse is not necessarily true. The stability of an otherwise unstable virus may result from the scarcity of an “active” phenol. For example, TAMV is highly senitive to oxidized chlorogenic acid and loses infectivity rapidly in tobacco leaf extracts where the primary phenol is chlorogenic acid (Zucker and Ahrens, 1958). Both AMV and PVX are insensitive to oxidized chlorogenie acid and are stable in tobacco juice. Except possibly for the report of Harrison and Pierpoint (1963)) all viruses that have been demonstrated to be inactivated by oxidized phenolic compounds appear to be inactivated by the o-benzoquinone structure rather than some transient intermediate This is substantiated by the fact that TCQ, which presumably participates only in oxidation-reduction reactions, can be used to produce predictable levels of inactivation. The results obtained here indicate that as the o-quinone structure becomes more complex (i.e., catechol, DOPA, anp chlorogenic acid), it becomes limited in the number of viruses it can inactivate. In previous tests when the molecular size of 4methyl-o-benzoquinone was increased by attachment to ovalbumin protein, that compound lost its ability to inactivate TAMV (Mink et al., 1966a). The available data continue to suggest that the steric configuration of the quinone and perhaps the structural integrity of virus proteins determine virus susceptibility to a particular o-quinone. However, it has not yet been established whether inactivation results from reactions between o-quinones and virus ribonucleic acid (RNA) or from a “tanning” of the virus protein. If RNA alone is in-
546
SAKSENA
volved it would appear that the permeability of virus proteins to low molecular weight compounds, such as quinones, varies considerably among viruses. REFERENCES J. G., and SAMUEL, G. (1934). Some factors affecting the inactivation rate of the virus of tomato spotted wilt. Ann. Appl. Biol. 21, 179190. BAWDEN, F. C., and PIRIE, N. W. (1957). A virus inactivating system from tobacco leaves. J. Gen. Microbial. 16,697-710. BENNETT, C. W., and COSTA, A. S. (1961). Sowbane mosaic caused by a seed-transmitted virus. Phytopathology 51,546-550. BEST, R. J. (1939). The preservative effect of some reducing systems on tomato spotted wilt virus. Sustralian J. Exptl. Biol. Med. Sci. 17, l-7. BRAKKE, M. K. (1953). Zonal separations by density-gradient centrifugation. Arch. Biochem. Biophys. 45, 275-290. FULTON, R. W. (1649). Virus concentration in plants acquiring tolerance to tobacco streak. Phytopathology 39, 231-243. FULTON, R. W. (1952). Mechanical transmission and properties of rose mosaic virus. Phytopathology 42, 413316. G~LVEZ, G. E. (1964). Loss of virus by filtration through charcoal. Virology 23, 307312. HAMPTON, R. E., and FULTON, R. W. (1961). The relation of polyphenol oxidase to instability in vitro of prune dwarf and sour cherry necrotic ringspot viruses. Virology 13, 4452. HARBORNE, J. B., ed. (1964). “Biochemistry of Phenolic Compounds.” Academic Press, New York. HARRISON, B. D., and PIERPOINT, W. S. (1963). The relation of polyphenoloxidase in leaf extracts to the instability of cucumber mosaic and other plant viruses. J. Gen. Microbial. 32, 417427. MARTIN, C. M. (1958). &ude cornparke de 1 activitb de la polyphdnoloxidase chew le tabac sain et inocule par une maladie B virus. Compt. Rend. Acad. Sci. 246, 2026-2029. BALD,
AND
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H. S. (1948). The chemistry of melanin. III. Mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. J. Biol. Chem. 172, 83-99. MASON, H. S. Q949). The chemistry of melanin. IV. Mechanism of the oxidat,ion of catechol by tyrosinase. J. Biol. Chem. 181,803~812. MASON, H. S. (1955). Comparative biochemistry of the phenolase complex. Advan. Enzymol. 16, 105-184. MINK, G. I. (1965). Inactivation of Tulare apple mosaic virus by o-quinones. Virology 26,700-707. MINK, G. I. (1967). Kinetics of quinone inactivation of Tulare apple mosaic virus. Virology 33, 609-612. MINK, G. I., BANCROFT, J. B., and NADAK.IVUKAREN, M. J. (1963). Properties of Tulare apple mosaic virus. Phytopathology 53, 973-978. MINK, G. I., HUISMAN, O., and SAKSENA, K. N. (1966a). Oxidative inactivation of Tulare apple mosaic virus. Virology 29, 437-443. MINK, G. I., SAKSENA, K. N., and SILBERNAGEL, M. J. (1966b). Purification of the bean red node strain of tobacco streak virus. Phytopathology 56, 645-649. Ross, A. F. (1941). Purification and properties of alfalfa-mosaic virus protein. Phytopathology 31, 394-410. SILBER, G., and BURK, L. G. (1965). Infectivity of tobacco mosaic virus stored for 50 years in extracted “unpreserved” plant juice. Nature 206, 740-741. STEERE, R. L. (1956). Purification and properties of tobacco ringspot virus. Phytopathology 46, 60-69. VAN KA~MEN, A., and BROUWER, D. (1964). Increase of polyphenol oxidase activity by a local virus infection in uninoculated parts of plants. Virology 22, 9-14. VASUDEVA, R. S., and L~L, T. B. (1945). Studies on the virus diseases of potatoes in India. I. Occurrence of Solanum virus 1. Indian J. ,4gr. Sci. 14, 28&295. ZUCKER, M., and AHRENS, J. F. (1958). Quantitative assay of chlorogenic acid and its pattern of distribution within tobacco leaves. Plant Physiol. 33, 246-249. MASON,