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The assembly of papaya mosaic virus protein
VIROLOGY
72,
514-517 (1976)
The Assembly
of Papaya Mosaic
J. W. ERICKSON
AND
Virus Protein
J. B. BANCROFT
Department of Plant Sciences, University of Western Ontario, London, Canada
R. W. HORNE John Znnes Institute, Norwich, England Accepted March 5,1976 Protein isolated from papaya mosaic virus, which is a flexuous virus similar in morphology to potato virus X, will self-assemble into long helical particles in the absence of RNA. The significance of the conditions required for assembly are briefly considered.
In a previous communication (l), we reported that coat protein isolated from the flexuous potato virus Y could be induced to form assembly products whose configuration differed from that of the virus. We now describe experiments with protein obtained from a shorter nonrigid tubular virus called papaya mosaic virus (PMV) (2). We show that protein from PMV will assemble into long viruslike helical particles in the absence of RNA. PMV was propagated in Carica papaya, and purified essentially as for PVX (3). Upper leaves from small (61-122 cm high) greenhouse-grown papaya trees infected for 6 to 8 months were harvested in 100-g lots and homogenized at 4” with 2 vol of 0.05 M, pH 8.0, sodium phosphate buffer containing 0.01 M EDTA. The homogenate was expressed through cheesecloth and clarified by centrifugation at 17,000 rpm for 20 min in a Sorvall centrifuge. The supernatant fluid was overlaid onto 7 ml of a 30% (w/v) sucrose solution containing 5 x 1O-3M EDTA, pH 7.6, and was centrifuged in a Beckman 30 rotor at 27,000 r-pm for 2V2 hr. The pellets were suspended in about 10 ml of 5 x 10e3M Tris, pH 7.6, or water. The supernatant fluid was then subjected to one to two additional cycles of differential centrifugation. Virus yields were 0.5 to 1.5 mg/g fresh weight tissue 514 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
and coat protein migrated as a single species CMW ca. 22,000) on SDS acrylamide gels. The virus yield could be doubled if the butanol-chloroform method was used as described by Purcifull and Hiebert (2). However, this method was not employed because products migrating faster than coat protein were sometimes observed in SDS gels in our hands, contrary to an earlier report (4). PMV protein was prepared by acetic acid degradation (5). Two vol of glacial acetic acid were added to 3 to 8 ml of virus at 5-15 mg/ml at 0 to 4”. The insoluble RNA was removed after 1 hr by centrifugation at 10,000 rpm for 15 min. The supernatant liquid was immediately centrifuged at 45,000 rpm for 4 hr in a Beckman 50 rotor to remove any residual virus. The solution was dialyzed for 48 hr against several changes of 0.01 M glycine, pH 3, or 0.01 M Tris, pH 8, depending on the experiment. The preparations were devoid of virus particles as judged by electron microscopy and gave typical protein spectra with A280/250 nm between 2.0 to 2.5..Protein concentrations were estimated assumingA!&$& = 1.0. Assembly experiments were performed by adjusting ionic conditions by 48 hour dialysis at 4” prior to examination at 20”. The starting protein was either at pH 3
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(0.01 M glycine) or pH 8 (0.01 M Tris). A single species sedimenting at 2.7 S probably corresponding to a dimer was found at pH 3 whereas the pH 8 material contained a series of small aggregates sedimenting between 13 and 33 S (Fig. 1A). Both types of starting preparation gave the same products upon further treatment.
515
At pH 4 (0.01 M citrate), polymers sedimenting at 13,100-120 and 230-250 S were formed (Fig. 1B). Such preparations were birefringent at 20” (polymerization is endothermic) and contained long particles (Fig. 2A). At pH 5 (0.01 M acetate) and 6 [O.Ol M 2-(N-morpholino)ethanesulfonate (MES)], the only product made was the 13-S species
FIG. 1. Schlieren diagrams of papaya mosaic virus protein after various treatments. (A) Upper: 13- and 33-S products in 0.01 M Tris, pH 8.0, after 13 min at 52,640 rpm; lower: 2.7-S species in 0.01 M giycine, pH 3. (B) 13,117- and 248-S products in 0.01 M citrate, pH 4.0, after 5 min at 29,500 rpm. (C) 13-S product in 0.01 M acetate, pH 5.0, after 31 min at 52,640 ‘pm. (D) 13-S product in 0.01 M MES, pH 6.0, after 31 min at 52,640 rpm. (E) 113- and 236-S products after dialysis of B vs 0.01 M acetate, pH 5, after 5 min at 29,500 rpm. Note the difference between (C) and (El. (F) 13- and 106-S products after dialysis of(E) versus 0.01 M MES, pH 6.0, after 6 min at 29,500. (G) 13- and 73-S products in 0.01 M citrate, pH 4,0.2 M NaCl after 25 min at 29,500 rpm. Note the difference between (B) and (G). (H) 13- and 101-S products in 0.01 M acetate, pH 5,0.2 M NaCl after 12 min at 29,500 rpm. The 101-S product has not been observed under the same centrifugal conditions in the absence of NaCl. (I) 13-S product in 0.01 M MES, pH 6, 0.2 M NaCl after 12 min at 29,500 rpm. Protein concentration was about 2 mg/ml at various bar angles. Sedimentation is to the right.
516
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FIG. 2. Electron micrographs and diffraction patterns of papaya mosaic virus protein and virus. (A) Photograph of material from Fig. ZB (X 36,000). (B) High resolution photograph (X 216,000) of the same material compared with (C) virus (x 107,200). (D) and (E) diffraction patterns of(B) and (0, respectively. The near meridional reflections correspond to a helical pitch of 36 A and the equatorial ones to a center-tocenter spacing of 130 A.
(Fig. lC, D). This may correspond to a double ring of subunits. A marked hysteresis effect was observed since protein initially assembled at pH 4 contained material which sedimented at 113 and 236 S after dialysis to pH 5 (Fig. 1E). This material was largely lost upon further dialysis to pH 6 (Fig. 1F). If the assembly experiments were performed in 0.2 M NaCl, the same general pattern of results as found at the lower ionic strength were obtained in that large numbers of sinuous particles
were found only at pH 4. However, these were not as long as observed in the absence of NaCl, the sedimentation coefficient being 73 S (Fig. 1G) and the particles being clearly shorter as seen in the electron microscope. At pH 5 and 6, the principal species sedimented at 13 S (Fig. lH, I), a small amount of faster sedimenting material (101 S) sometimes being found at pH 5. Electron micrographs for fine structure analysis were made of samples prepared
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by the method of Horne et al. (6, 7). Polymerized protein and native virus are shown in Fig. 2B and C, and the corresponding optical diffraction patterns in Fig. 2D, E. Both types of particles have a helical configuration with a pitch of 36 A and a diameter of about 130 A. We have not detected reflections suggestive of a double helix as recently described for polymers of narcissus mosaic virus protein (8). Although potato virus X protein has not yet been shown to assemble into helical arrays in the absence of RNA (9)) protein from PMV, which is a member of the same general group (101, will do so readily. These observations suggest that proteinprotein interactions play a more important role in virus stability for PMV than for potato virus X. Alternatively, they may derive from the conditions used for polymerization or from the methods employed in obtaining the protein. PMV, unlike PVX, will not dissociate in 2 it4 LiCl (II) or 2 M CaCl, 02) at pH 5 to 6. It will, with CaC1, at least, dissociate at pH 8, but the protein product is insoluble when the CaCl, is removed. The conditions necessary for the assembly of PMV protein differ widely from those required by protein from potato virus Y which, unlike PMV, forms long particles composed of packed rings or perhaps discs (1). It seems that proteins from different flexuous virus groups have different assembly requirements, as do proteins from rigid rod viruses such as TMV (13) and tobacco rattle virus (14). One basic structural difference between a rigid rod and a flexuous virus must lie in the radial distribution of axial interactions between coat protein subunits. Such geometric constraints do not necessarily rule on the specific chemical groups involved, nor do they relegate such groups to a virus with a particular shape. Thus, carboxyl-carboxylate pairs are critical in the control of assembly of protein from both TMV (15) and cowpea
chlorotic mottle virus (16) which is spherical. The pH levels required by PMV protein to make a helical structure suggest that a related mechanism involving acidic amino acid residues may also be operative in yet another group of viruses. ACKNOWLEDGMENTS We wish to thank Dr. J. G. McDonald for discussion, Dr. E. Hiebert for the original isolate of papaya mosaic virus and papaya seeds, and Mr. D. Muirhead for excellent technical assistance. This work was supported in part by the National Research Council of Canada. REFERENCES
9.
10.
11. 12.
13. 14. 15. 16.
MCDONALD, J. G., BEVERIDGE, T. J., and BANCROFT, J. B., Virology 69, 327-331 (1976). PURCIFULL, D. E., and HIEBERT, E., C.M.1.I A.A.B.‘No. 56 (1971). GOODMAN, R. M., Virology 68, 287-298 (1975). KOENIG, R., STEGEMANN, H., FRANCKSEN, H., and PAUL, H. L., Biochim. Biophys. Acta 207, 184-189 (1970). FRAENKEL-CONRAT, H., Virology 4,1-4 (1957). HORNE, R. W., and PA~QUALI-RONCHETTI, I., J. Ultrustruc. Res. 47, 361-383 (1974). HORNE, R. W., HOBART, J. M., and PA~QUALXRONCHETTI, I., Micron 5, 233-261 (1975). ROBINSON, D. J., HUTCHE~CIN, A., TOLLIN, P., and WILSON, H. R., J. Gen. Viral. 29,325-330 (1975). KAFTANOVA, A. S., KISELEV, N. A., NOVIKOV, V. K., and ATABEKOV, J. G., Virology 65,283-287 (1975). HARRISON, B. D., FINCH, J. T., GIBBS, A. J., HOLLINGS, M., SHEPHERD, R. J., VALENTO, V., and WETTER, C., Virology 45, 356-363 (1971). FRANCKI, R. I., and MCLEAN, G. D., Australian J. Biol. Sci. 21, 1311-1318 (1968). NOVIKOV, V. K., KIMAEV, V. Z., and ATABEKOV, J. G., Dokl. Akad. Nauk. SSSR 204, 12591262 (1972). DURHAM, A. C. H., and KLUG, A., Nature New Biol. 229, 42-46 (1971). FRITSCH, C., WITZ, J., ABOU-HAIDAR, M., and HIRTH, L., FEBS Lett. 29, 211-214 (1973). CASPAR, D. L. D., Adu. Protein Chem. 1837-121 (1963). BANCROFT, J. B., Adu. Virus Res. 16, 99-134 (1970).
72,
514-517 (1976)
The Assembly
of Papaya Mosaic
J. W. ERICKSON
AND
Virus Protein
J. B. BANCROFT
Department of Plant Sciences, University of Western Ontario, London, Canada
R. W. HORNE John Znnes Institute, Norwich, England Accepted March 5,1976 Protein isolated from papaya mosaic virus, which is a flexuous virus similar in morphology to potato virus X, will self-assemble into long helical particles in the absence of RNA. The significance of the conditions required for assembly are briefly considered.
In a previous communication (l), we reported that coat protein isolated from the flexuous potato virus Y could be induced to form assembly products whose configuration differed from that of the virus. We now describe experiments with protein obtained from a shorter nonrigid tubular virus called papaya mosaic virus (PMV) (2). We show that protein from PMV will assemble into long viruslike helical particles in the absence of RNA. PMV was propagated in Carica papaya, and purified essentially as for PVX (3). Upper leaves from small (61-122 cm high) greenhouse-grown papaya trees infected for 6 to 8 months were harvested in 100-g lots and homogenized at 4” with 2 vol of 0.05 M, pH 8.0, sodium phosphate buffer containing 0.01 M EDTA. The homogenate was expressed through cheesecloth and clarified by centrifugation at 17,000 rpm for 20 min in a Sorvall centrifuge. The supernatant fluid was overlaid onto 7 ml of a 30% (w/v) sucrose solution containing 5 x 1O-3M EDTA, pH 7.6, and was centrifuged in a Beckman 30 rotor at 27,000 r-pm for 2V2 hr. The pellets were suspended in about 10 ml of 5 x 10e3M Tris, pH 7.6, or water. The supernatant fluid was then subjected to one to two additional cycles of differential centrifugation. Virus yields were 0.5 to 1.5 mg/g fresh weight tissue 514 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
and coat protein migrated as a single species CMW ca. 22,000) on SDS acrylamide gels. The virus yield could be doubled if the butanol-chloroform method was used as described by Purcifull and Hiebert (2). However, this method was not employed because products migrating faster than coat protein were sometimes observed in SDS gels in our hands, contrary to an earlier report (4). PMV protein was prepared by acetic acid degradation (5). Two vol of glacial acetic acid were added to 3 to 8 ml of virus at 5-15 mg/ml at 0 to 4”. The insoluble RNA was removed after 1 hr by centrifugation at 10,000 rpm for 15 min. The supernatant liquid was immediately centrifuged at 45,000 rpm for 4 hr in a Beckman 50 rotor to remove any residual virus. The solution was dialyzed for 48 hr against several changes of 0.01 M glycine, pH 3, or 0.01 M Tris, pH 8, depending on the experiment. The preparations were devoid of virus particles as judged by electron microscopy and gave typical protein spectra with A280/250 nm between 2.0 to 2.5..Protein concentrations were estimated assumingA!&$& = 1.0. Assembly experiments were performed by adjusting ionic conditions by 48 hour dialysis at 4” prior to examination at 20”. The starting protein was either at pH 3
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COMMUNICATIONS
(0.01 M glycine) or pH 8 (0.01 M Tris). A single species sedimenting at 2.7 S probably corresponding to a dimer was found at pH 3 whereas the pH 8 material contained a series of small aggregates sedimenting between 13 and 33 S (Fig. 1A). Both types of starting preparation gave the same products upon further treatment.
515
At pH 4 (0.01 M citrate), polymers sedimenting at 13,100-120 and 230-250 S were formed (Fig. 1B). Such preparations were birefringent at 20” (polymerization is endothermic) and contained long particles (Fig. 2A). At pH 5 (0.01 M acetate) and 6 [O.Ol M 2-(N-morpholino)ethanesulfonate (MES)], the only product made was the 13-S species
FIG. 1. Schlieren diagrams of papaya mosaic virus protein after various treatments. (A) Upper: 13- and 33-S products in 0.01 M Tris, pH 8.0, after 13 min at 52,640 rpm; lower: 2.7-S species in 0.01 M giycine, pH 3. (B) 13,117- and 248-S products in 0.01 M citrate, pH 4.0, after 5 min at 29,500 rpm. (C) 13-S product in 0.01 M acetate, pH 5.0, after 31 min at 52,640 ‘pm. (D) 13-S product in 0.01 M MES, pH 6.0, after 31 min at 52,640 rpm. (E) 113- and 236-S products after dialysis of B vs 0.01 M acetate, pH 5, after 5 min at 29,500 rpm. Note the difference between (C) and (El. (F) 13- and 106-S products after dialysis of(E) versus 0.01 M MES, pH 6.0, after 6 min at 29,500. (G) 13- and 73-S products in 0.01 M citrate, pH 4,0.2 M NaCl after 25 min at 29,500 rpm. Note the difference between (B) and (G). (H) 13- and 101-S products in 0.01 M acetate, pH 5,0.2 M NaCl after 12 min at 29,500 rpm. The 101-S product has not been observed under the same centrifugal conditions in the absence of NaCl. (I) 13-S product in 0.01 M MES, pH 6, 0.2 M NaCl after 12 min at 29,500 rpm. Protein concentration was about 2 mg/ml at various bar angles. Sedimentation is to the right.
516
SHORT COMMUNICATIONS
FIG. 2. Electron micrographs and diffraction patterns of papaya mosaic virus protein and virus. (A) Photograph of material from Fig. ZB (X 36,000). (B) High resolution photograph (X 216,000) of the same material compared with (C) virus (x 107,200). (D) and (E) diffraction patterns of(B) and (0, respectively. The near meridional reflections correspond to a helical pitch of 36 A and the equatorial ones to a center-tocenter spacing of 130 A.
(Fig. lC, D). This may correspond to a double ring of subunits. A marked hysteresis effect was observed since protein initially assembled at pH 4 contained material which sedimented at 113 and 236 S after dialysis to pH 5 (Fig. 1E). This material was largely lost upon further dialysis to pH 6 (Fig. 1F). If the assembly experiments were performed in 0.2 M NaCl, the same general pattern of results as found at the lower ionic strength were obtained in that large numbers of sinuous particles
were found only at pH 4. However, these were not as long as observed in the absence of NaCl, the sedimentation coefficient being 73 S (Fig. 1G) and the particles being clearly shorter as seen in the electron microscope. At pH 5 and 6, the principal species sedimented at 13 S (Fig. lH, I), a small amount of faster sedimenting material (101 S) sometimes being found at pH 5. Electron micrographs for fine structure analysis were made of samples prepared
SHORT
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by the method of Horne et al. (6, 7). Polymerized protein and native virus are shown in Fig. 2B and C, and the corresponding optical diffraction patterns in Fig. 2D, E. Both types of particles have a helical configuration with a pitch of 36 A and a diameter of about 130 A. We have not detected reflections suggestive of a double helix as recently described for polymers of narcissus mosaic virus protein (8). Although potato virus X protein has not yet been shown to assemble into helical arrays in the absence of RNA (9)) protein from PMV, which is a member of the same general group (101, will do so readily. These observations suggest that proteinprotein interactions play a more important role in virus stability for PMV than for potato virus X. Alternatively, they may derive from the conditions used for polymerization or from the methods employed in obtaining the protein. PMV, unlike PVX, will not dissociate in 2 it4 LiCl (II) or 2 M CaCl, 02) at pH 5 to 6. It will, with CaC1, at least, dissociate at pH 8, but the protein product is insoluble when the CaCl, is removed. The conditions necessary for the assembly of PMV protein differ widely from those required by protein from potato virus Y which, unlike PMV, forms long particles composed of packed rings or perhaps discs (1). It seems that proteins from different flexuous virus groups have different assembly requirements, as do proteins from rigid rod viruses such as TMV (13) and tobacco rattle virus (14). One basic structural difference between a rigid rod and a flexuous virus must lie in the radial distribution of axial interactions between coat protein subunits. Such geometric constraints do not necessarily rule on the specific chemical groups involved, nor do they relegate such groups to a virus with a particular shape. Thus, carboxyl-carboxylate pairs are critical in the control of assembly of protein from both TMV (15) and cowpea
chlorotic mottle virus (16) which is spherical. The pH levels required by PMV protein to make a helical structure suggest that a related mechanism involving acidic amino acid residues may also be operative in yet another group of viruses. ACKNOWLEDGMENTS We wish to thank Dr. J. G. McDonald for discussion, Dr. E. Hiebert for the original isolate of papaya mosaic virus and papaya seeds, and Mr. D. Muirhead for excellent technical assistance. This work was supported in part by the National Research Council of Canada. REFERENCES
9.
10.
11. 12.
13. 14. 15. 16.
MCDONALD, J. G., BEVERIDGE, T. J., and BANCROFT, J. B., Virology 69, 327-331 (1976). PURCIFULL, D. E., and HIEBERT, E., C.M.1.I A.A.B.‘No. 56 (1971). GOODMAN, R. M., Virology 68, 287-298 (1975). KOENIG, R., STEGEMANN, H., FRANCKSEN, H., and PAUL, H. L., Biochim. Biophys. Acta 207, 184-189 (1970). FRAENKEL-CONRAT, H., Virology 4,1-4 (1957). HORNE, R. W., and PA~QUALI-RONCHETTI, I., J. Ultrustruc. Res. 47, 361-383 (1974). HORNE, R. W., HOBART, J. M., and PA~QUALXRONCHETTI, I., Micron 5, 233-261 (1975). ROBINSON, D. J., HUTCHE~CIN, A., TOLLIN, P., and WILSON, H. R., J. Gen. Viral. 29,325-330 (1975). KAFTANOVA, A. S., KISELEV, N. A., NOVIKOV, V. K., and ATABEKOV, J. G., Virology 65,283-287 (1975). HARRISON, B. D., FINCH, J. T., GIBBS, A. J., HOLLINGS, M., SHEPHERD, R. J., VALENTO, V., and WETTER, C., Virology 45, 356-363 (1971). FRANCKI, R. I., and MCLEAN, G. D., Australian J. Biol. Sci. 21, 1311-1318 (1968). NOVIKOV, V. K., KIMAEV, V. Z., and ATABEKOV, J. G., Dokl. Akad. Nauk. SSSR 204, 12591262 (1972). DURHAM, A. C. H., and KLUG, A., Nature New Biol. 229, 42-46 (1971). FRITSCH, C., WITZ, J., ABOU-HAIDAR, M., and HIRTH, L., FEBS Lett. 29, 211-214 (1973). CASPAR, D. L. D., Adu. Protein Chem. 1837-121 (1963). BANCROFT, J. B., Adu. Virus Res. 16, 99-134 (1970).