Proc. Natl. Acad. Sci. USA Vol. 74, No. 1, pp. 149-153, January 1977 Biochemistry
Inside-out model for self-assembly of tobacco mosaic virus (electron microscopy)
G. LEBEURIER, A. NICOLAIEFF, AND K. E. RICHARDS Laboratoire de Virologie, Institut de Biologie Moleculaire et Cellulaire du CNRS, Universite Louis Pasteur, 15, rue Descartes, 67000 Strasbourg, France
Communicated by Robley C. Williams, October 22, 1976
ABSTRACT Incompletely reconstituted particles of tobacco mosaic virus treated with moderate concentrations of dimethyl sulfoxide prior to electron microscopy have two visible tails of unencapsidated RNA, a long one and a short one. The short tail has a constant length of 720 i 80 nucleotides in incomplete particles of diverse size and probably corresponds to the 3'0H end of the RNA. The length of the long 5' tail is inversely related to the length of the unfinished rod. Surprisingly, both tails pear to protrude from the same end of the particle. These o servations suggest that the strand of RNA coming out of one end of the particle loops back along the length of the rod so as to appear at the opposite end. The "looped-back" tail almost certainly passes down the empty central channel of the incomplete rod. The findings are compatible with a model of tobacco mosaic virus assembly in which the RNA inserts itself beneath layers of incoming protein from within the central channel. Tobacco mosaic virus (TMV) is a hollow cylinder of length 300 nm, inner diameter 4 nm, and outer diameter 18 nm. Each particle contains 2200 protein subunits arranged as a right-hand helix with 16% subunits per turn. The RNA chain (6600 nucleotides) is sandwiched between successive turns of the protein helix. The self-assembly of TMV in vitro from its protein and RNA components is initiated by the binding of a bilayer disk of TMV protein to a site on the RNA molecule. Upon or immediately after binding, the protein subunits of the disk are thought to undergo a coordinated rearrangement to yield a helical nucleoprotein complex (lock-washer) which serves as the substrate for further addition of protein (1). Previous electron microscopic studies of TMV reconstitution, in which only initiation and a little rod growth were allowed to take place, showed a single "tail" of uncoated RNA protruding from one end of the incomplete pFitcle, leading to the idea that the origin of assembly was at or very near one end of the RNA chain (1-3). Several independent lines of evidence were marshalled in support of this conclusion and all seemed to point to the 5' end as the starting site (1, 3-5). However, more recent observations indicate that, contrary to the earlier findings, neither the 5' nor the 3' extremity of the RNA is encapsidated early in assembly (6, 7). Evidently, the apparent onetailed appearance of incompletely reconstituted particles deserves reinvestigation. In earlier publications we have described the use of dimethyl sulfoxide (Me2SO) to prepare single-stranded RNA as extended filaments for electron microscopy (8, 9) and to uncoat TMV particles (10, 11). In the latter studies single-stranded RNA could be perceived protruding from one or both ends of partially uncoated particles and the lengths of the tails could be measured. Hence we decided to use Me2SO as an aid in the visualization of the tails of partially reconstituted particles. In this paper we shall show that incompletely reconstituted particles treated with moderate concentrations of Me2S) have Abbreviations: TMV, tobacco mosaic virus; Me2SO, dimethyl sulfoxide. 149
two tails, one short and one much longer. Interestingly enough, both tails appear to protrude from the same end of the rod. This observation is most plausibly interpreted by the hypothesis that one of the RNA tails must traverse the length of the incomplete rod by passing along the central channel. This leads to a model for TMV assembly in which, during all but the last stages of reconstitution, the virus builds itself from the inside out, that is, the RNA inserts itself into the helix from within the central channel of the rod.
MATERIALS AND METHODS Partial Reconstitution of TMV. Incompletely reconstituted particles were prepared by reacting TMV RNA with a fourfold weight excess of 25S TMV disk protein in 0.5 IS sodium pyrophosphate, pH 7.25, for 20 min at 240 (12). The particles were collected by sedimentation for 3 hr at 100,000 X g in a Beckman 50 rotor. The pellet of incomplete particles was then gently dispersed in 0.01 M potassium phosphate, pH 7.0. Specimen Preparation. The sample solution to be spread by the Kleinschmidt technique (13) was prepared by adding 10 ,ul of partially reconstituted particles (1 ,g/ml with respect to RNA) to 200 ,gl of 60% Me2SO in water. Fifty microliters of cytochrome c (500 Ag/ml in 90% Me2SO) was then added and the solution was immediately spread on a distilled water hypophase (11). A portion of the protein film was picked up on a Siemens platinum grid covered by a carbon-Formvar film and washed a few seconds in water, then in 90% ethanol, and finally in 50 mM uranyl acetate in 90% ethanol. Excess liquid was removed by touching the edge of the grid with a piece of filter paper. Specimens were rotary shadowed with platinum, and micrographs were taken at a nominal magnification of 10,000X with a Siemens 101 electron microscope. The exact magnification was determined with a standard TMV preparation. Micrographs were magnified 20X with a Nikon Profile Projector and the RNA profile was traced. The contour length of the tracing was measured with a curvimeter. Observed tail length was converted to nucleotides by dividing by 0.32 nm per nucleotide
(9). RESULTS Electron Microscopy of Incompletely Reconstituted Particles. As noted in the introduction, there is now good evidence that, in spite of the previous reports to the contrary, incompletely reconstituted virus particles should have two RNA tails. If the site of initiation for reconstitution is quite near one end of the RNA, one of the two tails could simply be too short to be seen. Alternatively, the two tails could be held together by RNA-RNA interactions so as to appear as one thread of RNA in the electron microscope. In order to investigate this second possibility more fully, we treated incompletely assembled particles with Me2SO in an effort to dissociate the two tails from one another.
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FIG. 1.
Proc. Natl. Acad. Sci. USA 74 (1977)
(Legend appears at bottom of the following page.)
Biochemistry:
Lebeurier et al.
In preliminary experiments, TMV RNA was spread in the presence of various concentrations of Me2SO. It was found that the mean contour length of the RNA spread in 60% Me2SO (2.12 jum) was the same as that observed when the RNA was spread under more stringent denaturing conditions (90% Me2SO) (9). At the same time, length measurements of incompletely reconstituted particles spread with and without 60% Me2SO gave closely similar distributions of rod length. Thus, 60% Me2SO is sufficient to unravel most of the RNA secondary structure but is below the threshold of Me2SO concentration at which significant uncoating of the incomplete particles occurs. Fig. 1A shows a typical field from an electron micrograph of incompletely reconstituted particles treated with 60% Me2SO before spreading. A gallery of particle images is shown in Fig. 1B-D. It is evident that each incomplete particle has two visible tails, generally of dissimilar length. In every instance the twin tails appear to protrude from the same end of the rod. We have examined many such images and find that essentially all (>95%) of the incomplete particles in a typical preparation have such an appearance. When particles of different size are examined, the length of the longer RNA tail is inversely related to the length of the incomplete rod (Fig. 2A); the average length of the short tail, on the other hand, is independent of particle length (Fig. 2B). The implications of this latter observation will be explored more fully below. From time to time, incomplete particles were found which appeared to have more than two tails (Fig. IE). The extra tails may protrude from opposite ends of the rod, as in the example shown in Fig. 1, or all the tails may seem to come out of the same end. We attribute the supplementary tails to fortuitous binding of one or more molecules of free RNA to the end(s) of the incomplete rod. (The ends of such rods may be somewhat "sticky".) Although the many-tailed particles are a distinct minority, their existence raises the question of whether one of the two tails of the two-tailed particles that constitute the major species might arise by similar adventitious binding of free RNA. However, there are several arguments against this possibility; perhaps the most persuasive is that for the two-tailed particles, the sum of the lengths of the short tail and the long tail, when added to the length of the RNA already encapsidated, is usually close to the expected chain length for one molecule of TMV RNA (Fig. 2). Structural Model for Incompletely Reconstituted Rods. The above observations suggest that in an incompletely assembled rod the strand of RNA coming out of one end of the particle loops back along the length of the rod so as to appear to protrude from the opposite end. The "looped-back" tail could either run back along the outside of the rod or pass down the empty central channel. Against the former possibility is the fact that two-tailed particles produced by uncoating TMV with 74% Me2SO have the expected appearance, i.e., the two tails are at opposite ends of the rod (11). We take this to mean that in incompletely reconstituted particles, as opposed to. partially stripped rods, the second tail is firmly held in the looped-back configuration, as would be the case if it passes back along the central channel of the virus. Although this conclusion seems at first somewhat surprising, we shall show below that it is readily reconciled with other evidence, including recent x-ray crystallographic studies on the structure of the disk (14). Fig. 2B shows that the mean length of the short tail of incomplete rods corresponds to 720 ± 80 nucleotides. Zimmern and Wilson (personal communication) estimate that the site of
151
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E
8~~~~~ *a * .0 2000
z
B
SHORT TAIL
-J
400,00
2
PARTICLE LENGTH (nm)
FIG. 2. Length of the long RNA tail (A) and the short RNA tail (B) of incomplete particles as a function of nucleoprotein rod length. Fifty particles of diverse size were selected for measure. In 10 cases the sum of the length of the long and short tails plus the length of the RNA already encapsidated was less than or equal to 5600 nucleotides and presumably represent particles with a broken tail; in two cases the sum was greater than 7600 nucleotides. These particles are not included in the graph. (B) Dashed line is the relation expected between short tail length and particle size if the short rather than the long RNA tail passes down the central channel (see text).
initiation of TMV assembly is 900-1300 nucleotides from the 3'OH end of the RNA chain. Hence the short tail almost certainly corresponds to the 3' terminus of the RNA molecule. The fact that the short tail remains constant in length for incomplete particles ranging from 30 to 200 nm in size (Fig. 2B) indicates that, after initiation, addition of protein to the end of the nucleation complex containing the 5' tail is much favored over growth in the 3' direction. A plausible model for the structure of a short, incompletely reconstituted particle is shown in Fig. 3. In the model, it is the long 5' tail that threads back through the central channel. As the nucleoprotein rod grows, the 5' tail feeds up through the tube to be sandwiched between successive layers of newly added protein. The observed length of the short 3' tail remains unaffected by rod growth (at least during all but the later stages of assembly) and should correspond to the distance between the initiation site for assembly and the 3' end. The observed constancy in length of the short tail allows us to rule out the alternative model for incomplete particle structure, where it is the 3' tail rather than the 5' tail that passes along the hole in the rod. In such a model the 3' -5' polarity of elongation would require that rod growth take place at the end of the particle from which the twin tails protrude. As the 5' tail became covered with protein, more and more of the 3' tail would be imprisoned within the central channel of the growing rod. Hence, contrary to observation (Fig. 2), the short as well as the long tail should appear to diminish in length as classes of longer incomplete particles are considered..
FIG. 1 (on preceding page). Electron micrograph of incompletely reconstituted TMV particles treated with 60% Me2SO. (A) typical field, X68,000; (B-D) selected particles with the two-tailed aspect, X136,000; (E) rare "four-tailed monster" X136,000.
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Proc. Nati. Acad. Sci. USA 74 (1977)
4
ELONGATION
I INITIATION
3'
FIG. 3. Schematic diagram of the structure of an incompletely
reconstituted particle. The drawing is not to scale.
DISCUSSION The evidence presented in this paper favors a model of TMV assembly in which the RNA tail containing the-5' end passes back along the central channel, with protein adding to the growing rod at the end where the RNA turns back on itself. It is of interest to consider how in the course of assembly the 5' tail comes to be threaded down the central channel. A recent x-ray crystallographic investigation of the structure of the disk (14) shows that the two layers of the disk are held together by protein-protein contacts at high radius, but that at low radius the layers slew apart. Consequently, access to the RNA binding groove might well be easier from the inside of the disk than from the outside. If, during initiation of reconstitution, the RNA inserts itself between layers of the first disk from the inside, then the region of the RNA involved in initiation would be expected to have a loop structure since it is difficult to imagine how the RNA could enter the central hole of the disk without being folded back upon itself. Hence, it is gratifying that the sequence of the part of the RNA chain involved in nucleation of assembly can be cast into a stable hair-pin loop (Jonard and Richards, personal observations). It is readily seen that an initiation step in which a loop of RNA penetrates between the layers of the first disk from inside will inevitably lead to two-tailed particles of the observed aspect since one of the two arms of the initiating loop must remain in the central channel. The growing tip of the rod at which addition of protein takes place (Fig. 4) is admirably suited to accept further protein, either in the form of disks (1) or as smaller aggregates (15-17). The part of the RNA chain awaiting encapsidation lies extended down the central channel and so can swivel about its axis in the tube as new protein is added. In all of the foregoing discussion we have confined ourselves to consideration of rod growth along the long 5' tail. But eventually the 3' tail must be encapsidated as well if we are to have a complete particle. This appears to be a "late" event in assembly, since incomplete particles as long as 200 nm have undergone no detectable growth in that direction (Fig. 2B). It may be that the 5' tail protruding from the central channel hampers addition of protein back toward the 3' end of the RNA; encapsidation of the 3'-terminal tail could then occur only after the 5' tail has been pulled up out of the way by reconstitution. In infected tobacco cells the functional messenger for synthesis of TMV coat protein is a smaller RNA produced from the genome RNA by partial transcription or specific cleavage (18).
FIG. 4. The growing end of a short, incomplete, reconstituted
particle.
The coat protein messenger, known as the low-molecularweight component (19), corresponds to the 3'-terminal 750 nucleotides of the genome RNA (18). This is, of course, exactly the extent of the short 3' tail of incomplete particles. This circumstance suggests a possible mechanism for the control of coat protein production. As noted above, the initial burst of synthesis of coat protein in the infected cell must await the production of some low-molecular-weight component. Once some coat protein has been synthesized and disks have formed, reconstitution will begin. As shown in this paper, the portion of the TMV RNA containing the sequence for low-molecular-weight component will be the last part of the RNA to be coated. Hence, production of this component from the uncoated 3' tail of incomplete rods could continue (perhaps by partial replication), leading in turn to more coat protein for reconstitution. We acknowledge with gratitude the contribution of Prof. L. Hirth, who provided the initial impetus to perform the experiments reported here and subsequently made many useful suggestions. We thank Dr. B. Jacrot for many stimulating discussions, Dr. D. Zimmern for communication of results prior to publication, and Dr. A. C. H. Durham for reading the manuscript. 1. Butler, P. J. G. & Klug, A. (1971) "Assembly of the particle of tobacco mosaic virus from RNA and disk of protein," Nature New Biol. 229,47-50. 2. Stussi, C., Lebeurier, G. & Hirth, L. (1969) "Partial reconstitution of tobacco mosaic virus," Virology 38, 16-25.
Biochemistry: Lebeurier et al. 3. Ohno, T., Nozu, Y. & Okada, Y. (1971) "Polar reconstitution of tobacco mosaic virus," Virology 44, 510-516. 4. Thouvenel, J. C., Guilley, H., Stussi, C. & Hirth, L. (1971) "Evidence for polar reconstitution of TMV," FEBS Lett. 16, 204206. 5. Guilley, H., Stussi, C. & Hirth, L. (1971) "Influence de la phosphodiesterase de rate de porc sur la reconstitution in vitro du virus de la mosaique du tabac," C. R. HeW. Seances Acad. Sci. Ser. D 272, 1181-1184. 6. Zimmern, D. (1975) "The 5' end group of tobacco mosaic virus RNA is m7G5' ppp5' Gp," Nucleic Acids Res. 2, 1189-1201. 7. Zimmern, D. (1976) "The region of TMV RNA involved in the nucleation of assembly," Phil. Trans. R. Soc. London Ser. B, in press. 8. Oudet, P., Lebeurier, G. & Nicolaieff, A. (1970) "Etude du RNA du virus de la mosaique du tabac (VMT) par microscopie electronique," 7Ume Congres International de Microscopie electronique, Grenoble 1, 597. 9. Nicolaieff, A., Pinck, L., Koenig-Nikes, A. & Hirth, L. (1972) "Electron microscopy of replicative form and single strand RNA of alfalfa mosaic virus," J. Gen. Virol. 16, 47-59. 10. Nicolaieff, A., Lebeurier, G. & Hirth, L. (1974) "Electron microscopy and infectivity studies of the action of dimethylsulfoxide on two strains of tobacco mosaic virus," J. Gen. Virol. 22, 4353.
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11. Nicolaieff, A., Lebeurier, G., Morel, M. C. & Hirth, L. (1975) "The uncoating of native and reconstituted TMV by DMSO: the polarity of stripping," J. Gen. Virol. 26, 295-306. 12. Richards, K. E., Morel, M. C., Nicolaieff, A., Lebeurier, G. & Hirth, L. (1975) "Location of the cistron of the tobacco mosaic virus coat protein," Biochimie 57, 749-755. 13. Kleinschmidt, A. & Zahn, R. K. (1959) "Uber desoxyribonucleisauire-Molekulen in protein Mischfilmen," Zeitschrift fur Naturforschung 14b, 770-779. 14. Champness, J. N., Bloomer, A. C., Bricogne, G., Butler, P. J. G. & Klug, A. (1976) "The structure of the protein disk of tobacco mosaic virus to 5 A resolution," Nature 259, 20-24. 15. Richards, K. E. & Williams, R. C. (1973) "Assembly of tobacco mosaic virus rods in vitro. Elongation of partially assembly rods," Biochemistry 12, 4574-4581. 16. Okada, Y. (1975) "Mechanism of assembly of tobacco mosaic virus in vitro," Adv. Biophys. 7, 1-41. 17. Lebeurier, G. & Hirth, L. (1975) "Le virus de la mosaique du tabac: un modele de morphogenese virale in vitro," Bull. Inst. Pasteur, Paris 73,141-165. 18. Hunter, T. R., Hunt, T., Knowland, J. & Zimmern, D. (1976) "Messenger RNA for the coat protein of tobacco mosaic virus," Nature 260,759-764. 19. Siegel, A., Zaitlin, M. & Duda, C. T. (1973) "Replication of tobacco mosaic virus. IV. Further characterization of virus related RNAs," Virology 53,75-83.
Inside-out model for self-assembly of tobacco mosaic virus (electron microscopy)
G. LEBEURIER, A. NICOLAIEFF, AND K. E. RICHARDS Laboratoire de Virologie, Institut de Biologie Moleculaire et Cellulaire du CNRS, Universite Louis Pasteur, 15, rue Descartes, 67000 Strasbourg, France
Communicated by Robley C. Williams, October 22, 1976
ABSTRACT Incompletely reconstituted particles of tobacco mosaic virus treated with moderate concentrations of dimethyl sulfoxide prior to electron microscopy have two visible tails of unencapsidated RNA, a long one and a short one. The short tail has a constant length of 720 i 80 nucleotides in incomplete particles of diverse size and probably corresponds to the 3'0H end of the RNA. The length of the long 5' tail is inversely related to the length of the unfinished rod. Surprisingly, both tails pear to protrude from the same end of the particle. These o servations suggest that the strand of RNA coming out of one end of the particle loops back along the length of the rod so as to appear at the opposite end. The "looped-back" tail almost certainly passes down the empty central channel of the incomplete rod. The findings are compatible with a model of tobacco mosaic virus assembly in which the RNA inserts itself beneath layers of incoming protein from within the central channel. Tobacco mosaic virus (TMV) is a hollow cylinder of length 300 nm, inner diameter 4 nm, and outer diameter 18 nm. Each particle contains 2200 protein subunits arranged as a right-hand helix with 16% subunits per turn. The RNA chain (6600 nucleotides) is sandwiched between successive turns of the protein helix. The self-assembly of TMV in vitro from its protein and RNA components is initiated by the binding of a bilayer disk of TMV protein to a site on the RNA molecule. Upon or immediately after binding, the protein subunits of the disk are thought to undergo a coordinated rearrangement to yield a helical nucleoprotein complex (lock-washer) which serves as the substrate for further addition of protein (1). Previous electron microscopic studies of TMV reconstitution, in which only initiation and a little rod growth were allowed to take place, showed a single "tail" of uncoated RNA protruding from one end of the incomplete pFitcle, leading to the idea that the origin of assembly was at or very near one end of the RNA chain (1-3). Several independent lines of evidence were marshalled in support of this conclusion and all seemed to point to the 5' end as the starting site (1, 3-5). However, more recent observations indicate that, contrary to the earlier findings, neither the 5' nor the 3' extremity of the RNA is encapsidated early in assembly (6, 7). Evidently, the apparent onetailed appearance of incompletely reconstituted particles deserves reinvestigation. In earlier publications we have described the use of dimethyl sulfoxide (Me2SO) to prepare single-stranded RNA as extended filaments for electron microscopy (8, 9) and to uncoat TMV particles (10, 11). In the latter studies single-stranded RNA could be perceived protruding from one or both ends of partially uncoated particles and the lengths of the tails could be measured. Hence we decided to use Me2SO as an aid in the visualization of the tails of partially reconstituted particles. In this paper we shall show that incompletely reconstituted particles treated with moderate concentrations of Me2S) have Abbreviations: TMV, tobacco mosaic virus; Me2SO, dimethyl sulfoxide. 149
two tails, one short and one much longer. Interestingly enough, both tails appear to protrude from the same end of the rod. This observation is most plausibly interpreted by the hypothesis that one of the RNA tails must traverse the length of the incomplete rod by passing along the central channel. This leads to a model for TMV assembly in which, during all but the last stages of reconstitution, the virus builds itself from the inside out, that is, the RNA inserts itself into the helix from within the central channel of the rod.
MATERIALS AND METHODS Partial Reconstitution of TMV. Incompletely reconstituted particles were prepared by reacting TMV RNA with a fourfold weight excess of 25S TMV disk protein in 0.5 IS sodium pyrophosphate, pH 7.25, for 20 min at 240 (12). The particles were collected by sedimentation for 3 hr at 100,000 X g in a Beckman 50 rotor. The pellet of incomplete particles was then gently dispersed in 0.01 M potassium phosphate, pH 7.0. Specimen Preparation. The sample solution to be spread by the Kleinschmidt technique (13) was prepared by adding 10 ,ul of partially reconstituted particles (1 ,g/ml with respect to RNA) to 200 ,gl of 60% Me2SO in water. Fifty microliters of cytochrome c (500 Ag/ml in 90% Me2SO) was then added and the solution was immediately spread on a distilled water hypophase (11). A portion of the protein film was picked up on a Siemens platinum grid covered by a carbon-Formvar film and washed a few seconds in water, then in 90% ethanol, and finally in 50 mM uranyl acetate in 90% ethanol. Excess liquid was removed by touching the edge of the grid with a piece of filter paper. Specimens were rotary shadowed with platinum, and micrographs were taken at a nominal magnification of 10,000X with a Siemens 101 electron microscope. The exact magnification was determined with a standard TMV preparation. Micrographs were magnified 20X with a Nikon Profile Projector and the RNA profile was traced. The contour length of the tracing was measured with a curvimeter. Observed tail length was converted to nucleotides by dividing by 0.32 nm per nucleotide
(9). RESULTS Electron Microscopy of Incompletely Reconstituted Particles. As noted in the introduction, there is now good evidence that, in spite of the previous reports to the contrary, incompletely reconstituted virus particles should have two RNA tails. If the site of initiation for reconstitution is quite near one end of the RNA, one of the two tails could simply be too short to be seen. Alternatively, the two tails could be held together by RNA-RNA interactions so as to appear as one thread of RNA in the electron microscope. In order to investigate this second possibility more fully, we treated incompletely assembled particles with Me2SO in an effort to dissociate the two tails from one another.
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FIG. 1.
Proc. Natl. Acad. Sci. USA 74 (1977)
(Legend appears at bottom of the following page.)
Biochemistry:
Lebeurier et al.
In preliminary experiments, TMV RNA was spread in the presence of various concentrations of Me2SO. It was found that the mean contour length of the RNA spread in 60% Me2SO (2.12 jum) was the same as that observed when the RNA was spread under more stringent denaturing conditions (90% Me2SO) (9). At the same time, length measurements of incompletely reconstituted particles spread with and without 60% Me2SO gave closely similar distributions of rod length. Thus, 60% Me2SO is sufficient to unravel most of the RNA secondary structure but is below the threshold of Me2SO concentration at which significant uncoating of the incomplete particles occurs. Fig. 1A shows a typical field from an electron micrograph of incompletely reconstituted particles treated with 60% Me2SO before spreading. A gallery of particle images is shown in Fig. 1B-D. It is evident that each incomplete particle has two visible tails, generally of dissimilar length. In every instance the twin tails appear to protrude from the same end of the rod. We have examined many such images and find that essentially all (>95%) of the incomplete particles in a typical preparation have such an appearance. When particles of different size are examined, the length of the longer RNA tail is inversely related to the length of the incomplete rod (Fig. 2A); the average length of the short tail, on the other hand, is independent of particle length (Fig. 2B). The implications of this latter observation will be explored more fully below. From time to time, incomplete particles were found which appeared to have more than two tails (Fig. IE). The extra tails may protrude from opposite ends of the rod, as in the example shown in Fig. 1, or all the tails may seem to come out of the same end. We attribute the supplementary tails to fortuitous binding of one or more molecules of free RNA to the end(s) of the incomplete rod. (The ends of such rods may be somewhat "sticky".) Although the many-tailed particles are a distinct minority, their existence raises the question of whether one of the two tails of the two-tailed particles that constitute the major species might arise by similar adventitious binding of free RNA. However, there are several arguments against this possibility; perhaps the most persuasive is that for the two-tailed particles, the sum of the lengths of the short tail and the long tail, when added to the length of the RNA already encapsidated, is usually close to the expected chain length for one molecule of TMV RNA (Fig. 2). Structural Model for Incompletely Reconstituted Rods. The above observations suggest that in an incompletely assembled rod the strand of RNA coming out of one end of the particle loops back along the length of the rod so as to appear to protrude from the opposite end. The "looped-back" tail could either run back along the outside of the rod or pass down the empty central channel. Against the former possibility is the fact that two-tailed particles produced by uncoating TMV with 74% Me2SO have the expected appearance, i.e., the two tails are at opposite ends of the rod (11). We take this to mean that in incompletely reconstituted particles, as opposed to. partially stripped rods, the second tail is firmly held in the looped-back configuration, as would be the case if it passes back along the central channel of the virus. Although this conclusion seems at first somewhat surprising, we shall show below that it is readily reconciled with other evidence, including recent x-ray crystallographic studies on the structure of the disk (14). Fig. 2B shows that the mean length of the short tail of incomplete rods corresponds to 720 ± 80 nucleotides. Zimmern and Wilson (personal communication) estimate that the site of
151
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E
8~~~~~ *a * .0 2000
z
B
SHORT TAIL
-J
400,00
2
PARTICLE LENGTH (nm)
FIG. 2. Length of the long RNA tail (A) and the short RNA tail (B) of incomplete particles as a function of nucleoprotein rod length. Fifty particles of diverse size were selected for measure. In 10 cases the sum of the length of the long and short tails plus the length of the RNA already encapsidated was less than or equal to 5600 nucleotides and presumably represent particles with a broken tail; in two cases the sum was greater than 7600 nucleotides. These particles are not included in the graph. (B) Dashed line is the relation expected between short tail length and particle size if the short rather than the long RNA tail passes down the central channel (see text).
initiation of TMV assembly is 900-1300 nucleotides from the 3'OH end of the RNA chain. Hence the short tail almost certainly corresponds to the 3' terminus of the RNA molecule. The fact that the short tail remains constant in length for incomplete particles ranging from 30 to 200 nm in size (Fig. 2B) indicates that, after initiation, addition of protein to the end of the nucleation complex containing the 5' tail is much favored over growth in the 3' direction. A plausible model for the structure of a short, incompletely reconstituted particle is shown in Fig. 3. In the model, it is the long 5' tail that threads back through the central channel. As the nucleoprotein rod grows, the 5' tail feeds up through the tube to be sandwiched between successive layers of newly added protein. The observed length of the short 3' tail remains unaffected by rod growth (at least during all but the later stages of assembly) and should correspond to the distance between the initiation site for assembly and the 3' end. The observed constancy in length of the short tail allows us to rule out the alternative model for incomplete particle structure, where it is the 3' tail rather than the 5' tail that passes along the hole in the rod. In such a model the 3' -5' polarity of elongation would require that rod growth take place at the end of the particle from which the twin tails protrude. As the 5' tail became covered with protein, more and more of the 3' tail would be imprisoned within the central channel of the growing rod. Hence, contrary to observation (Fig. 2), the short as well as the long tail should appear to diminish in length as classes of longer incomplete particles are considered..
FIG. 1 (on preceding page). Electron micrograph of incompletely reconstituted TMV particles treated with 60% Me2SO. (A) typical field, X68,000; (B-D) selected particles with the two-tailed aspect, X136,000; (E) rare "four-tailed monster" X136,000.
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4
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I INITIATION
3'
FIG. 3. Schematic diagram of the structure of an incompletely
reconstituted particle. The drawing is not to scale.
DISCUSSION The evidence presented in this paper favors a model of TMV assembly in which the RNA tail containing the-5' end passes back along the central channel, with protein adding to the growing rod at the end where the RNA turns back on itself. It is of interest to consider how in the course of assembly the 5' tail comes to be threaded down the central channel. A recent x-ray crystallographic investigation of the structure of the disk (14) shows that the two layers of the disk are held together by protein-protein contacts at high radius, but that at low radius the layers slew apart. Consequently, access to the RNA binding groove might well be easier from the inside of the disk than from the outside. If, during initiation of reconstitution, the RNA inserts itself between layers of the first disk from the inside, then the region of the RNA involved in initiation would be expected to have a loop structure since it is difficult to imagine how the RNA could enter the central hole of the disk without being folded back upon itself. Hence, it is gratifying that the sequence of the part of the RNA chain involved in nucleation of assembly can be cast into a stable hair-pin loop (Jonard and Richards, personal observations). It is readily seen that an initiation step in which a loop of RNA penetrates between the layers of the first disk from inside will inevitably lead to two-tailed particles of the observed aspect since one of the two arms of the initiating loop must remain in the central channel. The growing tip of the rod at which addition of protein takes place (Fig. 4) is admirably suited to accept further protein, either in the form of disks (1) or as smaller aggregates (15-17). The part of the RNA chain awaiting encapsidation lies extended down the central channel and so can swivel about its axis in the tube as new protein is added. In all of the foregoing discussion we have confined ourselves to consideration of rod growth along the long 5' tail. But eventually the 3' tail must be encapsidated as well if we are to have a complete particle. This appears to be a "late" event in assembly, since incomplete particles as long as 200 nm have undergone no detectable growth in that direction (Fig. 2B). It may be that the 5' tail protruding from the central channel hampers addition of protein back toward the 3' end of the RNA; encapsidation of the 3'-terminal tail could then occur only after the 5' tail has been pulled up out of the way by reconstitution. In infected tobacco cells the functional messenger for synthesis of TMV coat protein is a smaller RNA produced from the genome RNA by partial transcription or specific cleavage (18).
FIG. 4. The growing end of a short, incomplete, reconstituted
particle.
The coat protein messenger, known as the low-molecularweight component (19), corresponds to the 3'-terminal 750 nucleotides of the genome RNA (18). This is, of course, exactly the extent of the short 3' tail of incomplete particles. This circumstance suggests a possible mechanism for the control of coat protein production. As noted above, the initial burst of synthesis of coat protein in the infected cell must await the production of some low-molecular-weight component. Once some coat protein has been synthesized and disks have formed, reconstitution will begin. As shown in this paper, the portion of the TMV RNA containing the sequence for low-molecular-weight component will be the last part of the RNA to be coated. Hence, production of this component from the uncoated 3' tail of incomplete rods could continue (perhaps by partial replication), leading in turn to more coat protein for reconstitution. We acknowledge with gratitude the contribution of Prof. L. Hirth, who provided the initial impetus to perform the experiments reported here and subsequently made many useful suggestions. We thank Dr. B. Jacrot for many stimulating discussions, Dr. D. Zimmern for communication of results prior to publication, and Dr. A. C. H. Durham for reading the manuscript. 1. Butler, P. J. G. & Klug, A. (1971) "Assembly of the particle of tobacco mosaic virus from RNA and disk of protein," Nature New Biol. 229,47-50. 2. Stussi, C., Lebeurier, G. & Hirth, L. (1969) "Partial reconstitution of tobacco mosaic virus," Virology 38, 16-25.
Biochemistry: Lebeurier et al. 3. Ohno, T., Nozu, Y. & Okada, Y. (1971) "Polar reconstitution of tobacco mosaic virus," Virology 44, 510-516. 4. Thouvenel, J. C., Guilley, H., Stussi, C. & Hirth, L. (1971) "Evidence for polar reconstitution of TMV," FEBS Lett. 16, 204206. 5. Guilley, H., Stussi, C. & Hirth, L. (1971) "Influence de la phosphodiesterase de rate de porc sur la reconstitution in vitro du virus de la mosaique du tabac," C. R. HeW. Seances Acad. Sci. Ser. D 272, 1181-1184. 6. Zimmern, D. (1975) "The 5' end group of tobacco mosaic virus RNA is m7G5' ppp5' Gp," Nucleic Acids Res. 2, 1189-1201. 7. Zimmern, D. (1976) "The region of TMV RNA involved in the nucleation of assembly," Phil. Trans. R. Soc. London Ser. B, in press. 8. Oudet, P., Lebeurier, G. & Nicolaieff, A. (1970) "Etude du RNA du virus de la mosaique du tabac (VMT) par microscopie electronique," 7Ume Congres International de Microscopie electronique, Grenoble 1, 597. 9. Nicolaieff, A., Pinck, L., Koenig-Nikes, A. & Hirth, L. (1972) "Electron microscopy of replicative form and single strand RNA of alfalfa mosaic virus," J. Gen. Virol. 16, 47-59. 10. Nicolaieff, A., Lebeurier, G. & Hirth, L. (1974) "Electron microscopy and infectivity studies of the action of dimethylsulfoxide on two strains of tobacco mosaic virus," J. Gen. Virol. 22, 4353.
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