- Email: [email protected]
Isolation and characterization of two temperature-sensitive mutants of cowpea mosaic virus
VIROLOGY
141,275-282
(1985)
Isolation and Characterization of Two Temperature-Sensitive Mutants of Cowpea Mosaic Virus D. EVANS’ John Inner Institute, Colneg Lane, Norwich NR4 7UH, England Received
September 4, 1984 accepted October 28, 1984
Two temperature-sensitive mutants of cowpea mosaic virus have been isolated following nitrous acid mutagenesis. One mutant, 8-14, was shown tn have a temperaturesensitive defect in the B component RNA. The location of the temperature-sensitive defect of the other mutant, 8-10, was not unambiguously determined. No virus-specific proteins were detected in leaves inoculated with the mutants and grown at the restrictive temperature. Dot blot analysis and infectivity assays indicated that reduced amounts of viral RNA were synthesized in leaves inoculated with 8-10 or 8-14 under restrictive o 19%Academic PWSS. I~C. conditions and the RNA was not encapsidated.
Wood (1972) isolated mutants of CPMV which showed altered local lesion morThe genome of cowpea mosaic virus phology on Phaseoh vulgaris var. Pinto (CPMV) consists of two separately encap- following nitrous acid mutagenesis. Gopo sidated molecules of RNA, the M-RNA and Frist (1977) were able to show that with 3841 nucleotides (van Wezenbeek et the coat proteins of CPMV are coded for aL, 1983) and the B-RNA with 5889 nuby the M-RNA by the characterization of cleotides (Lomonossoff and Shanks, 1983). a mutant induced by nitrous acid treatThe RNA is infectious, polyadenylated at ment of virions. De Jager and his cothe 3’ terminus and has a covalently linked workers have made an extensive genetic protein (VPg) at the 5’ terminus (Daubert study of CPMV mutants, isolating both et ak, 19’78;El Manna and Bruening, 1973; nitrous acid-induced and spontaneous Stanley et ab, 1978). mutants (de Jager, 1976; de Jager and During infection each RNA is first Breekland, 1979; de Jager and Wesseling, translated into a polyprotein which is 1981; de Jager et d, 1977). One of the subsequently cleaved to functional polymutants induced was temperature sensipeptides by virus-specific proteases (Dative and supplementation experiments vies et d, 1977; Goldbach et al, 1981; Pelham, 1979; Rezelman et al, 1980; Rot- showed that the mutation was located on tier et aL, 1980). The M-RNA codes for the M-RNA (de Jager et c& 1977). Although there is a considerable amount. the virus coat proteins (Frannsen et al, of information available on the structure 1982; Goldbach et aL, 1981; Gopo and of CPMV and the synthesis and processing Frist, 1977; Thongmeearkom and Goodof virus proteins, comparatively little is man, 1978), and the B-RNA codes for the known about viral functions such as unVPg, the virus-specific proteases and repcoating, encapsidation, cell-to-cell spread licase (Goldbach et d, 1980; Pelham, 1979; of virus, virus host range, assembly, and Stanley et u.L, 1980; Frannsen et 4, 1984, symptom induction. In order to identify Zabel et al, 1984). which virus gene(s) are involved in these phenomena, I have isolated a number of nitrous acid-induced mutants. This paper 1 Present address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AI,, reports the isolation of two temperatureEngland. sensitive mutants which produce reduced INTRODUCTION
275
0042~6822/85 Copyrixht All rights
$3.00
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
276
D. EVANS
amounts of RNA at the restrictive temperature. This RNA does not appear to be encapsidated. MATERIALS
AND
METHODS
Virus growth and pur$catim A stock of the Nigerian isolate of CPMV, obtained from Dr. C. P. de Jager was propagated in Vigna unguiculuta (L.) Walp. var. “Blackeye Early Ramshorn” and purified as described by van Kammen (196’7). Both wild-type and mutant stocks were maintained by local lesion transfer in Phaseolus vulgaris L. var. “Pinto.” The source of inoculum for all experiments was local lesions from “Pinto” leaves. Mutagenesis. Nitrous acid mutagenesis of virion RNA, purified by the method of Zimmern (1975), was exactly as described by de Jager et al. (1977). Temperature sensitivity. Primary leaves were detached from Vigna unguiculuta var. “Early Red” or var. “Blackeye Early Ramshorn” and opposite leaves were inoculated with a homogenate from a single lesion from “Pinto” leaves. The leaves were then placed on moist filter paper in petri dishes, one was incubated with continuous illumination at 20”, the other at 32”. In the case of “Early Red” the number of lesions visible after 6 days was determined. Samples of inoculated “Blackeye Early Ramshorn” leaves were removed 6-8 days postinoculation with a number 10 cork borer and were homogenized in 0.01 M phosphate buffer, pH 7. The amount of virus in the homogenate was determined by infectivity assays on “Pinto” leaves as described by de Jager (1976). Supplementation assays. Middle (M) and bottom (B) components were purified by two cycles of centrifugation in CsCl gradients at pH 8.5 (Stanley et al, 1978). The primary leaves were detached from either the “Early Red” or “Blackeye Early Ramshorn” varieties of Vigna unguiculata Opposite leaves were inoculated with a homogenate from a mutant lesion from “Pinto” which was supplemented with either M or B component from wild-type virus. The leaves were then placed on moist filter paper in petri dishes and
incubated with constant illumination at 32”. At 6-8 days postinoculation the amount of virus in homogenates of “Blackeye Early Ramshorn” leaves was determined by infectivity assay on Phaseolus vulgar& (de Jager, 1976). The number of necrotic lesions on “Early Red” leaves was counted 6 days postinoculation. Infected leaf proteins. Opposite primary leaves of “Blackeye Early Ramshorn” were detached and inoculated with a homogenate of a single lesion from a “Pinto” leaf inoculated with either wild-type or mutants 8-10 or 8-14. The leaves were placed on moist filter paper and incubated with constant illumination, one at 20’ and the other at 32”. At 8 days postinoculation samples of tissue were removed with a number 10 cork borer and were homogenized in 0.01 M phosphate buffer, pH 7. The homogenate was then centrifuged for 15 min in an Eppendorf microcentrifuge. A half volume of Laemmli gel sample buffer was added to the supernatant which was then boiled for 5 min. The samples were then analyzed on a 10% polyacrylamide gel (Laemmli, 1970). The proteins were visualized by silver staining (Oakley et al, 1980). “Dot blot” analysis of homogenutes of “Blackeye Early Ramshwn” leaves. Detached “Blackeye Early Ramshorn” leaves were inoculated with wild-type or mutant virus and incubated at 20 or 32” as described above. At 8 days postinoculation a homogenate of the inoculated leaves were prepared, applied directly to nitrocellulose, and baked (Kafatos et al., 1979). The “dot blots” were probed with nick-translated replicative form doublestranded DNA from Ml3 clones containing sequences specific to M- or B-RNA. The M clone consisted of the sequence from nucleotide residues 190-698, the B clone from nucleotide residues 2141-3979 (Lomonossoff and Shanks, 1983; Lomonossoff, personal communication). The clones were obtained from Dr. G. P. Lomonossoff, John Innes Institute. Dot blot analysis of RNA extracted from “Blackeye Early Ramshma” leaves. “Blackeye Early Ramshorn” leaves were inoculated with the mutants or wild-type
ISOLATION
OF ts MUTANTS
virus and incubated at 20 or 32” as described above. Eight days postinoculation the RNA was purified by treatment of extracts with phenol as described by Zelcer et al. (1981) and was analyzed on nitrocellulose as described above. RESULTS
of Mutants 8-10 and 8-14
Characterizatim
Following treatment with nitrous acid, the mutagenized RNA was used to inoculate Phaseolus vulgaris var. “Pinto.” Aberrant local lesions were selected. Two of these, designated 8-10 and 8-14, had phenotypes that were stable over a number of lesion transfers and were chosen for further study. Mutant 8-10 is characterized, on “Pinto,” by small necrotic lesions less than 1 mm in diameter. Mutant 8-14 induced large, diffuse chlorotic lesions when inoculated onto “Pinto” plants. Inoculation of “Pinto” leaves with the wildtype isolate of CPMV resulted in large, necrotic local lesions approximately 2 mm in diameter. The symptoms produced at 20 and 32” on “Blackeye Early Ramshorn” leaves following inoculation with the mutants are illustrated in Fig. 1. Neither mutant induced symptoms on inoculated leaves incubated at 32” whereas inoculation with
WILD
277
OF CPMV
wild-type virus and incubation at 32” resulted in extensive symptom development. The symptoms produced at 20” following inoculation with either mutant were similar to those induced by inoculation with wild-type virus. To quantify the amount of virus produced in leaves inoculated with the mutants at 20 and 32” infectivity assays of homogenized leaves were carried out on “Pinto” leaves (de Jager, 1976). The results obtained are shown in Table 1. Mutant 8-14 was completely temperature sensitive, no infectious virus being produced following incubation at 32”, the restrictive temperature. This temperature sensitivity probably was not due to thermolability of the virion since incubation of the inoculum at 32’ for 60 min before inoculation and subsequent incubation at 20” did not result in the loss of infectivity (data not shown). Mutant 8-10 was also temperature sensitive. At the restrictive temperature it produced only 7% of the virus produced at the permissive temperature. The virus that formed following inoculation under restrictive conditions with 8-10 was almost certainly due to reversion since out of 20 “Blackeye Early Ramshorn” leaves inoculated with 8-10 under restrictive conditions, infectious virus was detected in only five. When the temperature sensitiv-
TYPE
MOCK INOCULATED 32
20 FIG. 1. Infection of “Blackeye wild type, or mock inoculated.
Early
Ramshorn”
32
20
leaves at 20 or 32” with
mutants
8-10, 8-14,
278
D. EVANS TABLE 1
TEMPERATURE SENSITIVITY AND SUPPLEMENTATION WITH WILD-TYPE M OR B PARTICLES OF MUTANTS GROWN IN Vigna unguiculata VAR. “BLACKEYE EARLY RAMSHORN”
Number of lesions on Pinto half leaves’ Inoculum
20”
32”
32” + M
32” + B
8-10 8-14 Wild type M B M+B
142 133 330
10 0 124 0 6 133
116 12 136
202 132 136
“Numbers represent mean number of lesions per “Pinto” half leaf from at least 15 replicate experiments in which temperature sensitivity and supplementation assays were carried out.
ity of the virus produced at 32’ was determined the virus was not temperature sensitive. The temperature sensitivity of the mutants was also determined in the leaves of the variety “Early Red.” This assay has the advantage of being a direct determination, since the necrotic lesions can be counted on “Early Red” obviating the necessity for assaying in “Pinto.” The results are shown in Table 2 and confirm
TABLE 2 TEMPERATURE SENSITIVITY AND SUPPLEMENTATION WITH WILD-TYPE M OR B PARTICLES OF MUTANTS GROWN IN Vigna unguicuhta VAR. “EARLY RED”
Number of lesions per leaf” Inoculum
20’
32”
32” + M
32” + B
8-10 8-14 Wild type M B M+B
22 156 550
0 0 275 0 3 137
8 0
21 87
a Numbers represent mean from at least 10 replicate experiments in which temperature-sensitivity and supplementation assays were carried out.
those obtained using “Blackeye Early Ramshorn” as host. To determine on which component the lesion conferring temperature sensitivity lies, supplementation assays were carried out. The results obtained are shown in Tables 1 and 2. The mutation conferring temperature-sensitive growth on 8-14 is located on the B component RNA. The temperature sensitivity of 8-10 is apparently rescued by both M and B particles from wild-type virus. When “Blackeye Early Ramshorn” and “Early Red” leaves were doubly inoculated at the restrictive temperature with 8-10 and 8-14, no virus was produced (data not shown). This implies that these mutants were unable to complement and that the sites of the mutations were on the same component.
The proteins present in “Blackeye Early Ramshorn” leaves inoculated with wildtype virus, 8-10, 8-14, or mock inoculated and incubated at 20 or 32” are shown in Fig. 2. In leaves inoculated with wild type virus at 20”, three virus-specific bands could be detected. These are the two coat proteins, VP37 and VP23, and a smaller protein, VPZO, which is derived from VP23 by removal of the C-terminal amino acids (Kridl and Bruening, 1983). Inoculation with wild-type virus at 32” resulted in the synthesis of VP37. No VP23 was detected but VP20 was present. Inoculation with 8-10 at 20” resulted in a similar protein profile to that obtained with wildtype virus, whereas following inoculation at 32’ no virus proteins were detected. Following inoculation at 20° with 8-14, VP37 was detected, no VP23, and a small amount of VP20; at 32” no virus-specific proteins were detected. With this system the nonstructural proteins encoded by the B-RNA were not detected. Llcaction of Virus-Specific RNA Sew in IrLfected “Blackeye Early Rarnshorn ” Leaves
In order to determine whether the temperature-sensitive lesion was at the level
ISOLATION
OF ts MUTANTS
123456769101112
FIG. 2. Soluble proteins from “Blackeye Early Ramshorn” leaves infected with 8-10, 8-14 or wild type, or mock inoculated, at 20 or 32”. Analysis was by poiyacrylamide gel eiectrophoresis. Lane 1: molecular-weight markers; 2: mock inoculated, 20”; 3: mock inoculated, 32”; 4: wild type, 20’; 5: wild type, 32’; 6: 8-10, 20’; 7: 8-10, 32’; 8: 8-14, 20’; 9: 8-14, 32’; 10: mock inoculated, 20’; 11: mock inoculated, 32’; 12: molecular-weight markers. Numbers represent molecular weight X 1Om3.
of RNA synthesis, infected leaf homogenates were blotted onto nitrocellulose and probed with cDNA clones. Homogenates prepared from leaves infected at 20” with wild type, 8-10, or 8-14 all contained RNA sequences which hybridised to clones specific to both M- and B-RNA (Fig. 3). At 32” wild-type infected leaves produced approximately 25% of the viral RNA produced at 20”. No virus-specific RNA was detected in leaf homogenates derived from leaves infected at 32” with 8-10 or 8-14. In an attempt to quantitate the amount of virus-specific RNA produced, known amounts of virus RNA were added to mock-infected homogenates and analyzed as described above. However, under these conditions no virus specific RNA was detected, implying that the added RNA had been degraded. On the other hand, virusspecific RNA species were detected after addition of purified virus, showing that encapsidated RNA is protected from degradation. These experiments raised the
279
OF CPMV
possibility that the analysis of crude homogenates described above had only measured RNA which had been encapsidated. It is possible that in leaves inoculated with mutant virus under restrictive conditions viral RNA is produced but not encapsidated. To test this hypothesis “Blackeye” leaves were inoculated at 20 and 32” with wild type, 8-10, or 8-14. At 8 days postinoculation the leaves were frozen in liquid nitrogen, and the RNA was phenol extracted immediately. In contrast to the result obtained with total leaf homogenate, slightly more viral RNA sequences were detected in leaves inoculated with wild-type virus at 32” than in those infected at 20” (Fig. 4). Some virus-specific RNA was also detected in “Blackeye” leaves inoculated at 32” with 8-10 or 8-14, although in reduced amounts. Infectivity assays confirmed that no infectious virus was produced. This result suggests that under restrictive conditions some viral-specific RNA sequences are synthesized by the mutants but that they were not encapsidated and thus were degraded in extracts that were not treated with phenol.
8
Al
20
.3I
20
32
20
32
20
32
FIG. 3. Dot blot analysis of homogenates of “Blackeye Early Ramshorn” leaves infected with 8-10, 8-14, or wild type at 20 or 32”. Dilutions for each pair of dot columns of extract are, left to right, 1/ 2, l/5, l/10, l/20.
280
D. EVANS
B PROBE
M PROBE
20
32
20
32
20
32
20
32
FIG. 4. Dot blot analysis of RNA from phenol extracts of “Blackeye Early Ramshorn” leaves infected at 20 or 32” with 8-10, 8-14, or wild type. Dilutions for each pair of dot columns of extract are, left to right, l/l, l/2, l/5, l/10. DISCUSSION
The location of the ti defect of mutant 8-10 is not known. Supplementation with either wild-type M or B components allowed growth of S-10 at the restrictive temperature (32”). It is unlikely to be a double temperature-sensitive mutant with a lesion on each RNA since if this was the case supplementation with neither wild-type component would allow multiplication at the restrictive temperature. The finding that double inoculation with 8-10 and 8-14 at the restrictive temperature did not result in virus production implies that the mutation lies on the B-RNA since this is unambiguously the site of the temperature-sensitive defect of 8-14. If the mutation lies on the B component its effect on virus multiplication may be through an interaction with the M gene products. Thus supplementation with the wild-type M component would increase its gene dosage and thus allow some growth of virus under restrictive conditions. It should be noted that the level of virus production at 32” is considerably lower upon supplementation with wild-type M than with wild-type B component. Another explanation is that the purified wild-type components are not free from contamination with the other component. If this is the case it would imply
that the defect lies on the M component. This is because inoculation with M alone never resulted in virus production, whereas inoculation with B alone did result in very low levels of infectivity. The purified B component preparation must therefore contain a small amount of contaminating M component. In the case of mutant 8-14 the site of the ts defect was located on the B-RNA. The ts defect of a previously reported mutant of CPMV was located on the M component RNA (de Jager et aL, 1977). The B-RNA has been shown to code for the virus-coded RNA polymerase, the VPg and two proteases (Frannsen et al, 1984; Zabel et aL, 1984). The dot blot experiment indicates that the virus RNA sequences were produced following inoculation at the restrictive temperature with 8-10 and 8-14. However, further experimentation is required to determine the size and polarity of the transcripts and thus determine if there is a defect in the polymerase, or in some other factor that influences metabolism of the virus RNAs, resulting in defective RNA synthesis. However, since the RNA that was produced was not incorporated into infectious particles, the defect also could be in one of the proteases, which would result in defective processing of the M-RNA encoded polyprotein, or in the VPg, which may have a role in encap-
ISOLATION
OF ik MUTANTS
sidation. Experiments on in vitro protein processing at the permissive and restrictive temperatures should help discriminate between these possibilities. Direct sequencing of the protease and VPg coding regions of mutant and revertants would allow exact localization of the site of mutation. Mapping of a ts defect by direct sequencing has recently been achieved for a ts mutant of tobacco mosaic virus (Zimmern and Hunter, 1983). ACKNOWLEDGMENTS I thank Dr. G. P. Lomonossoff for much helpful advice and comment and for critically reading the manuscript. I also thank Dr. C. P. de Jager for advice on mutant isolation techniques and for the provision of virus stocks. I thank Dr. J. W. Davies for provision of facilities in his department and for his advice and encouragement. During the course of this work I was in receipt of a John Innes Charity Fellowship.
REFERENCES
DAUBERT,S.D., BRUENING,G.,and NAJARIAN,R.
C. (19’78). Protein bound to the genome RNAs of cowpea mosaic virus. Eur. J. Biochem 92.45-51. DAVIES, J. W., AALBERS, A. M. J., STUICK, E. J., and VAN KAMMEN, A. (1977). Translation of cowpea mosaic virus RNA in a cell-free extract from wheat germ. FEBS Z&t. 77, 265-269. DE JAGER, C. P. (1976). Genetic analysis of cowpea mosaic virus mutants by supplementation and reassortment tests. Virology 70. 151-163. DE JAGER, C. P., and BREEKLAND, L. (1979). Evidence for intrastrand complementation in cowpea mosaic virus infection. Virology 99, 312-318. DE JAGER, C. P., and WESSELING, J. B. M. (1981). Spontaneous mutations in cowpea mosaic virus overcoming resistance due to hypersensitivity in cowpea. Physiol Plant Pathol. 19, 34’7-358. DE JAGER, C. P., ZABEL, P., VAN DERBEEK, C. P., and VAN KAMMEN, A. (1977). Genetic and physiological characterization of a temperature-sensitive mutant of cowpea mosaic virus. Virology 76, 164-172. EL MANNA, M. M., and BRUENING, G. (1973). Polyadenylate sequences in the ribonucleic acids of cowpea mosaic virus. Virology 56, 198-206. FRANNSEN, H., GOLDBACH, R., BROEKHUISSEN, M., MOERMAN, M., and VAN KAMMEN, A. (1982). Expression of middle-component RNA of cowpea mosaic virus: In vitro generation of a precursor to both capsid proteins by a bottom-component RNA-
OF CPMV
281
encoded protease from infected cells. J. Vzrol. 41, 8-17. FRANNSEN, H., LEUNISSEN, J., GOLDBACH, R., LOMONOSSOFF, G., and ZIMMERN, D. (1984). Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses. EMBO J. 3,855861.
GOLDBACH,R.,REZELMAN, G.,and VAN KAMMEN, A. (1980). Independent replication and expression of B-component RNA of cowpea mosaic virus. Nature (London) 286, 297-300. GOLDBACH,R. W.,SCHILTHUIS, J.G., and REZELMAN, G. (1981). Comparison of in viva and in vitro translation of cowpea mosaic virus RNAs. Biochem Biophys. Res. Commun 99, 89-94. GOPO, J. M., and FRIST, R. H. (1977). Location of the gene specifying the smaller protein of the cowpea mosaic virus capsid. Virology 79, 259-266. KAFATOS,F. C., WELDON JONES,C., and EFSTRATIADIS, A. (1979). Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucl. Acids Rea 7, 15411552. KRIDL, J. C., and BRUENING, G. (1983). Comparison of capsids and nucleocapsids from cowpea mosaic virus-infected cowpea protoplasts and seedlings. Virology 129, 369-380. LAEMMLI, U. K. (1970). Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LOMONOSSOFF, G. P., and SHANKS, M. (1983). The nucleotide sequence of cowpea mosaic virus B RNA. EMBO .I 2, 2253-2258. OAKLEY, B. R., KIRSCH, D. R., and MORRIS, R. N. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105,361-363. PELHAM, H. R. B. (1979). Synthesis and proteolytic processing of cowpea mosaic virus proteins in reticulocyte Iysates. Virology 96, 463-477. REZELMAN,G., GOLDBACH,R.,and VAN KAMMEN, A. (1980). Expression of bottom component RNA of cowpea mosaic virus in cowpea protoplasts. J. Viral. 36, 366-371. ROTTIER, P. J. M., REZELMAN, G., and VAN KAMMEN, A. (1980). Protein synthesis in cowpea mosaic virus infected cowpea protoplasts: Detection of virus related proteins. J. Gen Viral. 51, 359-371. STANLEY, J., ROTTIER, P., DAVIES, J. W., ZABEL, P., and VAN KAMMEN, A. (1978). A protein linked to the 5’ termini of both RNA components of the cowpea mosaic virus genome. Nucl. Acio!s Res. 5, 4505-4522. STANLEY, J., GOLDBACH, R., and VAN KAMMEN, A. (1980). The genome-linked protein of cowpea mosaic virus is coded by RNA from the bottom component. Virology 106, 180-182.
282
D. EVANS
THONGMEEARKOM, P., and GOODMAN,R. M. (1978). Complementation and pseudorecombination between ribonucleie acids from two natural isolates of cowpea mosaic virus (severe subgroup). virology 85.75-83.
VAN KAYMEN, A. (1967). Purification and properties of the components of cowpea mosaic virus. virdogy 31, 633-642.
VAN WEZENBEEK,P., VERVER,J., HARMSEN,J., Vos, P., and VAN KAMMEN, A. (1983). Primary structure and gene organization of the middle component RNA of cowpea mosaic virus. EMBO J 2,941-946. WOOD, H. A, (1972). Genetic complementation between the two nucleoprotein components of cowpea mosaic virus. Viro!.ogjj 49, 592-598. ZABEL, P., MOERMAN, M., LOMONOSSOFF,G. P.,
SHANKS,M., and BEYREUTHER,K. (1984). Cowpea mosaic virus VPg: Sequencing of radiochemically modified protein allows mapping of the gene on B RNA. EMBO J. 3, 1629-1634. ZELCER,A., WEABER,K. F., BALAZS,E., and ZAITLIN, M. (1981). The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants. Vh?og~ 113, 417427.
ZIMMERN. D. (1975). The 5’ end group of tobacco mosaic virus RNA is m’G$ppaGp. Nucl Acids Res. 2, 1189-1201. ZIMMERN,D., and HUNTER,T. (1983). Point mutation in the 30K open reading frame of TMV implicated in temperature-sensitive assembly and local lesion spreading of mutant Ni2519. EMBO J. 2, 18931900.
141,275-282
(1985)
Isolation and Characterization of Two Temperature-Sensitive Mutants of Cowpea Mosaic Virus D. EVANS’ John Inner Institute, Colneg Lane, Norwich NR4 7UH, England Received
September 4, 1984 accepted October 28, 1984
Two temperature-sensitive mutants of cowpea mosaic virus have been isolated following nitrous acid mutagenesis. One mutant, 8-14, was shown tn have a temperaturesensitive defect in the B component RNA. The location of the temperature-sensitive defect of the other mutant, 8-10, was not unambiguously determined. No virus-specific proteins were detected in leaves inoculated with the mutants and grown at the restrictive temperature. Dot blot analysis and infectivity assays indicated that reduced amounts of viral RNA were synthesized in leaves inoculated with 8-10 or 8-14 under restrictive o 19%Academic PWSS. I~C. conditions and the RNA was not encapsidated.
Wood (1972) isolated mutants of CPMV which showed altered local lesion morThe genome of cowpea mosaic virus phology on Phaseoh vulgaris var. Pinto (CPMV) consists of two separately encap- following nitrous acid mutagenesis. Gopo sidated molecules of RNA, the M-RNA and Frist (1977) were able to show that with 3841 nucleotides (van Wezenbeek et the coat proteins of CPMV are coded for aL, 1983) and the B-RNA with 5889 nuby the M-RNA by the characterization of cleotides (Lomonossoff and Shanks, 1983). a mutant induced by nitrous acid treatThe RNA is infectious, polyadenylated at ment of virions. De Jager and his cothe 3’ terminus and has a covalently linked workers have made an extensive genetic protein (VPg) at the 5’ terminus (Daubert study of CPMV mutants, isolating both et ak, 19’78;El Manna and Bruening, 1973; nitrous acid-induced and spontaneous Stanley et ab, 1978). mutants (de Jager, 1976; de Jager and During infection each RNA is first Breekland, 1979; de Jager and Wesseling, translated into a polyprotein which is 1981; de Jager et d, 1977). One of the subsequently cleaved to functional polymutants induced was temperature sensipeptides by virus-specific proteases (Dative and supplementation experiments vies et d, 1977; Goldbach et al, 1981; Pelham, 1979; Rezelman et al, 1980; Rot- showed that the mutation was located on tier et aL, 1980). The M-RNA codes for the M-RNA (de Jager et c& 1977). Although there is a considerable amount. the virus coat proteins (Frannsen et al, of information available on the structure 1982; Goldbach et aL, 1981; Gopo and of CPMV and the synthesis and processing Frist, 1977; Thongmeearkom and Goodof virus proteins, comparatively little is man, 1978), and the B-RNA codes for the known about viral functions such as unVPg, the virus-specific proteases and repcoating, encapsidation, cell-to-cell spread licase (Goldbach et d, 1980; Pelham, 1979; of virus, virus host range, assembly, and Stanley et u.L, 1980; Frannsen et 4, 1984, symptom induction. In order to identify Zabel et al, 1984). which virus gene(s) are involved in these phenomena, I have isolated a number of nitrous acid-induced mutants. This paper 1 Present address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AI,, reports the isolation of two temperatureEngland. sensitive mutants which produce reduced INTRODUCTION
275
0042~6822/85 Copyrixht All rights
$3.00
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
276
D. EVANS
amounts of RNA at the restrictive temperature. This RNA does not appear to be encapsidated. MATERIALS
AND
METHODS
Virus growth and pur$catim A stock of the Nigerian isolate of CPMV, obtained from Dr. C. P. de Jager was propagated in Vigna unguiculuta (L.) Walp. var. “Blackeye Early Ramshorn” and purified as described by van Kammen (196’7). Both wild-type and mutant stocks were maintained by local lesion transfer in Phaseolus vulgaris L. var. “Pinto.” The source of inoculum for all experiments was local lesions from “Pinto” leaves. Mutagenesis. Nitrous acid mutagenesis of virion RNA, purified by the method of Zimmern (1975), was exactly as described by de Jager et al. (1977). Temperature sensitivity. Primary leaves were detached from Vigna unguiculuta var. “Early Red” or var. “Blackeye Early Ramshorn” and opposite leaves were inoculated with a homogenate from a single lesion from “Pinto” leaves. The leaves were then placed on moist filter paper in petri dishes, one was incubated with continuous illumination at 20”, the other at 32”. In the case of “Early Red” the number of lesions visible after 6 days was determined. Samples of inoculated “Blackeye Early Ramshorn” leaves were removed 6-8 days postinoculation with a number 10 cork borer and were homogenized in 0.01 M phosphate buffer, pH 7. The amount of virus in the homogenate was determined by infectivity assays on “Pinto” leaves as described by de Jager (1976). Supplementation assays. Middle (M) and bottom (B) components were purified by two cycles of centrifugation in CsCl gradients at pH 8.5 (Stanley et al, 1978). The primary leaves were detached from either the “Early Red” or “Blackeye Early Ramshorn” varieties of Vigna unguiculata Opposite leaves were inoculated with a homogenate from a mutant lesion from “Pinto” which was supplemented with either M or B component from wild-type virus. The leaves were then placed on moist filter paper in petri dishes and
incubated with constant illumination at 32”. At 6-8 days postinoculation the amount of virus in homogenates of “Blackeye Early Ramshorn” leaves was determined by infectivity assay on Phaseolus vulgar& (de Jager, 1976). The number of necrotic lesions on “Early Red” leaves was counted 6 days postinoculation. Infected leaf proteins. Opposite primary leaves of “Blackeye Early Ramshorn” were detached and inoculated with a homogenate of a single lesion from a “Pinto” leaf inoculated with either wild-type or mutants 8-10 or 8-14. The leaves were placed on moist filter paper and incubated with constant illumination, one at 20’ and the other at 32”. At 8 days postinoculation samples of tissue were removed with a number 10 cork borer and were homogenized in 0.01 M phosphate buffer, pH 7. The homogenate was then centrifuged for 15 min in an Eppendorf microcentrifuge. A half volume of Laemmli gel sample buffer was added to the supernatant which was then boiled for 5 min. The samples were then analyzed on a 10% polyacrylamide gel (Laemmli, 1970). The proteins were visualized by silver staining (Oakley et al, 1980). “Dot blot” analysis of homogenutes of “Blackeye Early Ramshwn” leaves. Detached “Blackeye Early Ramshorn” leaves were inoculated with wild-type or mutant virus and incubated at 20 or 32” as described above. At 8 days postinoculation a homogenate of the inoculated leaves were prepared, applied directly to nitrocellulose, and baked (Kafatos et al., 1979). The “dot blots” were probed with nick-translated replicative form doublestranded DNA from Ml3 clones containing sequences specific to M- or B-RNA. The M clone consisted of the sequence from nucleotide residues 190-698, the B clone from nucleotide residues 2141-3979 (Lomonossoff and Shanks, 1983; Lomonossoff, personal communication). The clones were obtained from Dr. G. P. Lomonossoff, John Innes Institute. Dot blot analysis of RNA extracted from “Blackeye Early Ramshma” leaves. “Blackeye Early Ramshorn” leaves were inoculated with the mutants or wild-type
ISOLATION
OF ts MUTANTS
virus and incubated at 20 or 32” as described above. Eight days postinoculation the RNA was purified by treatment of extracts with phenol as described by Zelcer et al. (1981) and was analyzed on nitrocellulose as described above. RESULTS
of Mutants 8-10 and 8-14
Characterizatim
Following treatment with nitrous acid, the mutagenized RNA was used to inoculate Phaseolus vulgaris var. “Pinto.” Aberrant local lesions were selected. Two of these, designated 8-10 and 8-14, had phenotypes that were stable over a number of lesion transfers and were chosen for further study. Mutant 8-10 is characterized, on “Pinto,” by small necrotic lesions less than 1 mm in diameter. Mutant 8-14 induced large, diffuse chlorotic lesions when inoculated onto “Pinto” plants. Inoculation of “Pinto” leaves with the wildtype isolate of CPMV resulted in large, necrotic local lesions approximately 2 mm in diameter. The symptoms produced at 20 and 32” on “Blackeye Early Ramshorn” leaves following inoculation with the mutants are illustrated in Fig. 1. Neither mutant induced symptoms on inoculated leaves incubated at 32” whereas inoculation with
WILD
277
OF CPMV
wild-type virus and incubation at 32” resulted in extensive symptom development. The symptoms produced at 20” following inoculation with either mutant were similar to those induced by inoculation with wild-type virus. To quantify the amount of virus produced in leaves inoculated with the mutants at 20 and 32” infectivity assays of homogenized leaves were carried out on “Pinto” leaves (de Jager, 1976). The results obtained are shown in Table 1. Mutant 8-14 was completely temperature sensitive, no infectious virus being produced following incubation at 32”, the restrictive temperature. This temperature sensitivity probably was not due to thermolability of the virion since incubation of the inoculum at 32’ for 60 min before inoculation and subsequent incubation at 20” did not result in the loss of infectivity (data not shown). Mutant 8-10 was also temperature sensitive. At the restrictive temperature it produced only 7% of the virus produced at the permissive temperature. The virus that formed following inoculation under restrictive conditions with 8-10 was almost certainly due to reversion since out of 20 “Blackeye Early Ramshorn” leaves inoculated with 8-10 under restrictive conditions, infectious virus was detected in only five. When the temperature sensitiv-
TYPE
MOCK INOCULATED 32
20 FIG. 1. Infection of “Blackeye wild type, or mock inoculated.
Early
Ramshorn”
32
20
leaves at 20 or 32” with
mutants
8-10, 8-14,
278
D. EVANS TABLE 1
TEMPERATURE SENSITIVITY AND SUPPLEMENTATION WITH WILD-TYPE M OR B PARTICLES OF MUTANTS GROWN IN Vigna unguiculata VAR. “BLACKEYE EARLY RAMSHORN”
Number of lesions on Pinto half leaves’ Inoculum
20”
32”
32” + M
32” + B
8-10 8-14 Wild type M B M+B
142 133 330
10 0 124 0 6 133
116 12 136
202 132 136
“Numbers represent mean number of lesions per “Pinto” half leaf from at least 15 replicate experiments in which temperature sensitivity and supplementation assays were carried out.
ity of the virus produced at 32’ was determined the virus was not temperature sensitive. The temperature sensitivity of the mutants was also determined in the leaves of the variety “Early Red.” This assay has the advantage of being a direct determination, since the necrotic lesions can be counted on “Early Red” obviating the necessity for assaying in “Pinto.” The results are shown in Table 2 and confirm
TABLE 2 TEMPERATURE SENSITIVITY AND SUPPLEMENTATION WITH WILD-TYPE M OR B PARTICLES OF MUTANTS GROWN IN Vigna unguicuhta VAR. “EARLY RED”
Number of lesions per leaf” Inoculum
20’
32”
32” + M
32” + B
8-10 8-14 Wild type M B M+B
22 156 550
0 0 275 0 3 137
8 0
21 87
a Numbers represent mean from at least 10 replicate experiments in which temperature-sensitivity and supplementation assays were carried out.
those obtained using “Blackeye Early Ramshorn” as host. To determine on which component the lesion conferring temperature sensitivity lies, supplementation assays were carried out. The results obtained are shown in Tables 1 and 2. The mutation conferring temperature-sensitive growth on 8-14 is located on the B component RNA. The temperature sensitivity of 8-10 is apparently rescued by both M and B particles from wild-type virus. When “Blackeye Early Ramshorn” and “Early Red” leaves were doubly inoculated at the restrictive temperature with 8-10 and 8-14, no virus was produced (data not shown). This implies that these mutants were unable to complement and that the sites of the mutations were on the same component.
The proteins present in “Blackeye Early Ramshorn” leaves inoculated with wildtype virus, 8-10, 8-14, or mock inoculated and incubated at 20 or 32” are shown in Fig. 2. In leaves inoculated with wild type virus at 20”, three virus-specific bands could be detected. These are the two coat proteins, VP37 and VP23, and a smaller protein, VPZO, which is derived from VP23 by removal of the C-terminal amino acids (Kridl and Bruening, 1983). Inoculation with wild-type virus at 32” resulted in the synthesis of VP37. No VP23 was detected but VP20 was present. Inoculation with 8-10 at 20” resulted in a similar protein profile to that obtained with wildtype virus, whereas following inoculation at 32’ no virus proteins were detected. Following inoculation at 20° with 8-14, VP37 was detected, no VP23, and a small amount of VP20; at 32” no virus-specific proteins were detected. With this system the nonstructural proteins encoded by the B-RNA were not detected. Llcaction of Virus-Specific RNA Sew in IrLfected “Blackeye Early Rarnshorn ” Leaves
In order to determine whether the temperature-sensitive lesion was at the level
ISOLATION
OF ts MUTANTS
123456769101112
FIG. 2. Soluble proteins from “Blackeye Early Ramshorn” leaves infected with 8-10, 8-14 or wild type, or mock inoculated, at 20 or 32”. Analysis was by poiyacrylamide gel eiectrophoresis. Lane 1: molecular-weight markers; 2: mock inoculated, 20”; 3: mock inoculated, 32”; 4: wild type, 20’; 5: wild type, 32’; 6: 8-10, 20’; 7: 8-10, 32’; 8: 8-14, 20’; 9: 8-14, 32’; 10: mock inoculated, 20’; 11: mock inoculated, 32’; 12: molecular-weight markers. Numbers represent molecular weight X 1Om3.
of RNA synthesis, infected leaf homogenates were blotted onto nitrocellulose and probed with cDNA clones. Homogenates prepared from leaves infected at 20” with wild type, 8-10, or 8-14 all contained RNA sequences which hybridised to clones specific to both M- and B-RNA (Fig. 3). At 32” wild-type infected leaves produced approximately 25% of the viral RNA produced at 20”. No virus-specific RNA was detected in leaf homogenates derived from leaves infected at 32” with 8-10 or 8-14. In an attempt to quantitate the amount of virus-specific RNA produced, known amounts of virus RNA were added to mock-infected homogenates and analyzed as described above. However, under these conditions no virus specific RNA was detected, implying that the added RNA had been degraded. On the other hand, virusspecific RNA species were detected after addition of purified virus, showing that encapsidated RNA is protected from degradation. These experiments raised the
279
OF CPMV
possibility that the analysis of crude homogenates described above had only measured RNA which had been encapsidated. It is possible that in leaves inoculated with mutant virus under restrictive conditions viral RNA is produced but not encapsidated. To test this hypothesis “Blackeye” leaves were inoculated at 20 and 32” with wild type, 8-10, or 8-14. At 8 days postinoculation the leaves were frozen in liquid nitrogen, and the RNA was phenol extracted immediately. In contrast to the result obtained with total leaf homogenate, slightly more viral RNA sequences were detected in leaves inoculated with wild-type virus at 32” than in those infected at 20” (Fig. 4). Some virus-specific RNA was also detected in “Blackeye” leaves inoculated at 32” with 8-10 or 8-14, although in reduced amounts. Infectivity assays confirmed that no infectious virus was produced. This result suggests that under restrictive conditions some viral-specific RNA sequences are synthesized by the mutants but that they were not encapsidated and thus were degraded in extracts that were not treated with phenol.
8
Al
20
.3I
20
32
20
32
20
32
FIG. 3. Dot blot analysis of homogenates of “Blackeye Early Ramshorn” leaves infected with 8-10, 8-14, or wild type at 20 or 32”. Dilutions for each pair of dot columns of extract are, left to right, 1/ 2, l/5, l/10, l/20.
280
D. EVANS
B PROBE
M PROBE
20
32
20
32
20
32
20
32
FIG. 4. Dot blot analysis of RNA from phenol extracts of “Blackeye Early Ramshorn” leaves infected at 20 or 32” with 8-10, 8-14, or wild type. Dilutions for each pair of dot columns of extract are, left to right, l/l, l/2, l/5, l/10. DISCUSSION
The location of the ti defect of mutant 8-10 is not known. Supplementation with either wild-type M or B components allowed growth of S-10 at the restrictive temperature (32”). It is unlikely to be a double temperature-sensitive mutant with a lesion on each RNA since if this was the case supplementation with neither wild-type component would allow multiplication at the restrictive temperature. The finding that double inoculation with 8-10 and 8-14 at the restrictive temperature did not result in virus production implies that the mutation lies on the B-RNA since this is unambiguously the site of the temperature-sensitive defect of 8-14. If the mutation lies on the B component its effect on virus multiplication may be through an interaction with the M gene products. Thus supplementation with the wild-type M component would increase its gene dosage and thus allow some growth of virus under restrictive conditions. It should be noted that the level of virus production at 32” is considerably lower upon supplementation with wild-type M than with wild-type B component. Another explanation is that the purified wild-type components are not free from contamination with the other component. If this is the case it would imply
that the defect lies on the M component. This is because inoculation with M alone never resulted in virus production, whereas inoculation with B alone did result in very low levels of infectivity. The purified B component preparation must therefore contain a small amount of contaminating M component. In the case of mutant 8-14 the site of the ts defect was located on the B-RNA. The ts defect of a previously reported mutant of CPMV was located on the M component RNA (de Jager et aL, 1977). The B-RNA has been shown to code for the virus-coded RNA polymerase, the VPg and two proteases (Frannsen et al, 1984; Zabel et aL, 1984). The dot blot experiment indicates that the virus RNA sequences were produced following inoculation at the restrictive temperature with 8-10 and 8-14. However, further experimentation is required to determine the size and polarity of the transcripts and thus determine if there is a defect in the polymerase, or in some other factor that influences metabolism of the virus RNAs, resulting in defective RNA synthesis. However, since the RNA that was produced was not incorporated into infectious particles, the defect also could be in one of the proteases, which would result in defective processing of the M-RNA encoded polyprotein, or in the VPg, which may have a role in encap-
ISOLATION
OF ik MUTANTS
sidation. Experiments on in vitro protein processing at the permissive and restrictive temperatures should help discriminate between these possibilities. Direct sequencing of the protease and VPg coding regions of mutant and revertants would allow exact localization of the site of mutation. Mapping of a ts defect by direct sequencing has recently been achieved for a ts mutant of tobacco mosaic virus (Zimmern and Hunter, 1983). ACKNOWLEDGMENTS I thank Dr. G. P. Lomonossoff for much helpful advice and comment and for critically reading the manuscript. I also thank Dr. C. P. de Jager for advice on mutant isolation techniques and for the provision of virus stocks. I thank Dr. J. W. Davies for provision of facilities in his department and for his advice and encouragement. During the course of this work I was in receipt of a John Innes Charity Fellowship.
REFERENCES
DAUBERT,S.D., BRUENING,G.,and NAJARIAN,R.
C. (19’78). Protein bound to the genome RNAs of cowpea mosaic virus. Eur. J. Biochem 92.45-51. DAVIES, J. W., AALBERS, A. M. J., STUICK, E. J., and VAN KAMMEN, A. (1977). Translation of cowpea mosaic virus RNA in a cell-free extract from wheat germ. FEBS Z&t. 77, 265-269. DE JAGER, C. P. (1976). Genetic analysis of cowpea mosaic virus mutants by supplementation and reassortment tests. Virology 70. 151-163. DE JAGER, C. P., and BREEKLAND, L. (1979). Evidence for intrastrand complementation in cowpea mosaic virus infection. Virology 99, 312-318. DE JAGER, C. P., and WESSELING, J. B. M. (1981). Spontaneous mutations in cowpea mosaic virus overcoming resistance due to hypersensitivity in cowpea. Physiol Plant Pathol. 19, 34’7-358. DE JAGER, C. P., ZABEL, P., VAN DERBEEK, C. P., and VAN KAMMEN, A. (1977). Genetic and physiological characterization of a temperature-sensitive mutant of cowpea mosaic virus. Virology 76, 164-172. EL MANNA, M. M., and BRUENING, G. (1973). Polyadenylate sequences in the ribonucleic acids of cowpea mosaic virus. Virology 56, 198-206. FRANNSEN, H., GOLDBACH, R., BROEKHUISSEN, M., MOERMAN, M., and VAN KAMMEN, A. (1982). Expression of middle-component RNA of cowpea mosaic virus: In vitro generation of a precursor to both capsid proteins by a bottom-component RNA-
OF CPMV
281
encoded protease from infected cells. J. Vzrol. 41, 8-17. FRANNSEN, H., LEUNISSEN, J., GOLDBACH, R., LOMONOSSOFF, G., and ZIMMERN, D. (1984). Homologous sequences in non-structural proteins from cowpea mosaic virus and picornaviruses. EMBO J. 3,855861.
GOLDBACH,R.,REZELMAN, G.,and VAN KAMMEN, A. (1980). Independent replication and expression of B-component RNA of cowpea mosaic virus. Nature (London) 286, 297-300. GOLDBACH,R. W.,SCHILTHUIS, J.G., and REZELMAN, G. (1981). Comparison of in viva and in vitro translation of cowpea mosaic virus RNAs. Biochem Biophys. Res. Commun 99, 89-94. GOPO, J. M., and FRIST, R. H. (1977). Location of the gene specifying the smaller protein of the cowpea mosaic virus capsid. Virology 79, 259-266. KAFATOS,F. C., WELDON JONES,C., and EFSTRATIADIS, A. (1979). Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucl. Acids Rea 7, 15411552. KRIDL, J. C., and BRUENING, G. (1983). Comparison of capsids and nucleocapsids from cowpea mosaic virus-infected cowpea protoplasts and seedlings. Virology 129, 369-380. LAEMMLI, U. K. (1970). Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LOMONOSSOFF, G. P., and SHANKS, M. (1983). The nucleotide sequence of cowpea mosaic virus B RNA. EMBO .I 2, 2253-2258. OAKLEY, B. R., KIRSCH, D. R., and MORRIS, R. N. (1980). A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105,361-363. PELHAM, H. R. B. (1979). Synthesis and proteolytic processing of cowpea mosaic virus proteins in reticulocyte Iysates. Virology 96, 463-477. REZELMAN,G., GOLDBACH,R.,and VAN KAMMEN, A. (1980). Expression of bottom component RNA of cowpea mosaic virus in cowpea protoplasts. J. Viral. 36, 366-371. ROTTIER, P. J. M., REZELMAN, G., and VAN KAMMEN, A. (1980). Protein synthesis in cowpea mosaic virus infected cowpea protoplasts: Detection of virus related proteins. J. Gen Viral. 51, 359-371. STANLEY, J., ROTTIER, P., DAVIES, J. W., ZABEL, P., and VAN KAMMEN, A. (1978). A protein linked to the 5’ termini of both RNA components of the cowpea mosaic virus genome. Nucl. Acio!s Res. 5, 4505-4522. STANLEY, J., GOLDBACH, R., and VAN KAMMEN, A. (1980). The genome-linked protein of cowpea mosaic virus is coded by RNA from the bottom component. Virology 106, 180-182.
282
D. EVANS
THONGMEEARKOM, P., and GOODMAN,R. M. (1978). Complementation and pseudorecombination between ribonucleie acids from two natural isolates of cowpea mosaic virus (severe subgroup). virology 85.75-83.
VAN KAYMEN, A. (1967). Purification and properties of the components of cowpea mosaic virus. virdogy 31, 633-642.
VAN WEZENBEEK,P., VERVER,J., HARMSEN,J., Vos, P., and VAN KAMMEN, A. (1983). Primary structure and gene organization of the middle component RNA of cowpea mosaic virus. EMBO J 2,941-946. WOOD, H. A, (1972). Genetic complementation between the two nucleoprotein components of cowpea mosaic virus. Viro!.ogjj 49, 592-598. ZABEL, P., MOERMAN, M., LOMONOSSOFF,G. P.,
SHANKS,M., and BEYREUTHER,K. (1984). Cowpea mosaic virus VPg: Sequencing of radiochemically modified protein allows mapping of the gene on B RNA. EMBO J. 3, 1629-1634. ZELCER,A., WEABER,K. F., BALAZS,E., and ZAITLIN, M. (1981). The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants. Vh?og~ 113, 417427.
ZIMMERN. D. (1975). The 5’ end group of tobacco mosaic virus RNA is m’G$ppaGp. Nucl Acids Res. 2, 1189-1201. ZIMMERN,D., and HUNTER,T. (1983). Point mutation in the 30K open reading frame of TMV implicated in temperature-sensitive assembly and local lesion spreading of mutant Ni2519. EMBO J. 2, 18931900.