Assembly of tobacco mosaic virus.

The assembly of tobacco mosaic virus requires the presence of a particular protein aggregate, the disk. During the nucleation, a specific region of the RNA interacts with a single disk, to bring about a necessarily cooperative transition from the paired two-layer structure to a short segment of nucleo-protein helix. There is a high selectivity for this region of the TMV RNA, because of the many nucleotides bound at once, and other nucleotide sequences appear only to bind by a different mechanism. Elongation of the nucleated rods can continue with either further disks or the less aggregated 'A-protein' as the protein source, but the continued cooperativity inherent with disks would have some advantages. The rates of the two processes have been separately determined and growth is faster when disks are still present. New experiments show that the breakdown of disks to yield A-protein is relatively slow and it is concluded that virus growth from disks could not proceed through a prior breakdown in solution, but must involve the direct interaction of the disk with the growing nucleoprotein rod. The detailed mechanism of disk addition is not understood but it may involve a directed breakdown, since there is also evidence for the existence of a non-equilibrium form of A-protein which has aggregation kinetics distinct from those of equilibrium A-protein. Some implications for the general assembly pathways of viruses both of the specificity and of the assembly/disassembly cycle during the viral infection are considered.     

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.     

Quantized incorporation of RNA during assembly of tobacco mosaic virus from protein disks

Reassembly of tobacco mosaic virus from the isolated RNA and protein, supplied as a disk preparation consisting of over 75% as the disk aggregate, has been followed by the protection of the RNA from nuclease digestion. The sizes of the RNA fragments were determined on agarose/acrylamide gels.During the first few minutes the protected RNA is found to be “quantized” into discrete lengths, differing on average by about 50 or 100 nucleotides, corresponding to one or two turns of the virus helix and strongly supporting the hypothesis that elongation in the major direction, towards the 5′-hydroxyl end, is occurring by the direct addition of protein disks. Protected RNA of the full length found in tobacco mosaic virus is visible within six minutes of starting reassembly, and by 30 minutes most of the RNA is fully protected.     

Effect of state of polymerisation of the protein component on the assembly of tobacco mosaic virus

Summary We had proposed that both the initiation and growth of tobacco mosaic virus rods takes place from the RNA and protein disks, containing 34 protein subunits. Other workers have reported that growth occurs not from disks but from A-protein. We now review their experiments and show that they have not used disks, but rather two-disk stacks and that their results, but not conclusions, are compatible with our earlier findings.     

Structures and roles of the polymorphic forms of tobacco mosaic virus protein. 8. Initial stages of assembly of nucleoprotein rods from virus RNA and the protein disks

The initial stages of the assembly of tobacco mosaic virus have been investigated by reassembling the RNA with a radioactively labelled protein disk preparation and then completing the reaction by the addition of a large excess of an unlabelled disk preparation. This gives measurements of the numbers of subunits incorporated at early times and the growth curves have been plotted.These curves have been analysed in terms of a bimolecular nucleation reaction, which is first order in the disk concentration, with a rate constant of 1.3 × 10³ mol^?1 s^?1, and then an elongation which saturates at high protein concentrations to a maximum rate of 7.6 subunits s^?1, with a K_m of 0.84 mg/ml for the disk preparation.These kinetic parameters, and the predicted overall assembly curves, have been compared with data previously determined by other methods and agree closely, showing that the different experimental techniques give consistent results. The measurements are fully compatible with our earlier hypotheses Butler &; Klug 1971 that the nucleation with virus RNA and protein disks is rapid compared with the subsequent rod elongation and that this elongation can occur most rapidly directly from the protein disks. They are not compatible with the contention of some other workers that elongation cannot occur directly from disks, but only from the smaller A-protein.     

RNA-protein interactions in the assembly of tobacco mosaic virus.

Assembly of tobacco mosaic virus is initiated by the binding of a specific loop of the RNA into the central hole of the disk aggregate of protein subunits. Since the nucleation loop is located about five-sixths along the RNA molecule, subsequent elongation must be bidirectional. We have now measured the rates of elongation in the two directions by determining the lengths of RNA protected from nuclease digestion at different times and using either intact TMV rNA, or RNA with most of the longer tail removed. Comparison of the rates with the protein supplied as either a mixture of disks with A-protein (a mixture of less aggregated states) or just A-protein, shows that different mechanisms and protein aggregates are used for the most rapid growth. When disks are present, they add more rapidly along the longer RNA tail but do not appear to add directly on the shorter tail. In contrast, smaller aggregates (A-protein) can add at both ends of the rod, but do so more slowly. Mechanisms for these processes are discussed. Preliminary results on the binding of the specific hexanucleotide AAGAAG to the disk are given and compared with the known changes on binding nonspecific hexanucleotides or the trinucleotide AAG.     

Self-assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed

The tobacco mosaic virus (TMV) particle was the first macromolecular structure to be shown to self-assemble in vitro, allowing detailed studies of the mechanism. Nucleation of TMV self-assembly is by the binding of a specific stem-loop of the single-stranded viral RNA into the central hole of a two-ring sub-assembly of the coat protein, known as the 'disk'. Binding of the loop onto its specific binding site, between the two rings of the disk, leads to melting of the stem so more RNA is available to bind. The interaction of the RNA with the protein subunits in the disk cause this to dislocate into a proto-helix, rearranging the protein subunits in such a way that the axial gap between the rings at inner radii closes, entrapping the RNA. Assembly starts at an internal site on TMV RNA, about 1 kb from its 3'-terminus, and the elongation in the two directions is different. Elongation of the nucleated rods towards the 5'-terminus occurs on a 'travelling loop' of the RNA and, predominantly, still uses the disk sub-assembly of protein subunits, consequently incorporating approximately 100 further nucleotides as each disk is added, while elongation towards the 3'-terminus uses smaller protein aggregates and does not show this 'quantized' incorporation.     

Structures and roles of the polymorphic forms of tobacco mosaic virus protein. VII. Lengths of the growing rods during assembly into nucleoprotein with the viral RNA

The length distributions of growing particles have been determined and followed as a function of time during the reconstitution of tobacco mosaic virus from its isolated RNA and protein. The protein was supplied either largely as the “disk” aggregate or as A-protein obtained by cooling a disk preparation. In a further experiment, the growth was initiated with disks and then continued with A-protein. It has been possible to correct the resulting distributions of lengths for the effect of broken RNA molecules and hence to obtain a picture of the distribution of lengths of the growing particles.From these distributions and also the average lengths, it is concluded that the growth is most rapid when disks are the protein source, giving full length particles in six to ten minutes. When A-protein is supplied for the growth, the rate is about one quarter of that with disks, irrespective of whether the rods have been nucleated with disks or not.     

The hand of the stacked-disk aggregate of tobacco mosaic virus protein

The hand of the helical arrangement of subunits in the stacked-disk form of tobacco mosaic virus protein has been determined from electron microscopy. This and previous structural results taken together indicate that the near-axial intersubunit contacts between disks are similar to those within a single disk but quite different from those in the virus. Discrepancies between the structures of single disks and stacked disks are attributed to cleavage of the protein within the stacked-disk rod.     

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.     

X-ray analysis of the disk of tobacco mosaic virus protein. II. The packing arrangement in the crystal

By a combined X-ray and electron microscopic analysis, the asymmetric unit of the crystal of tobacco mosaic virus protein has been identified with the disk aggregate, of molecular weight 600,000, composed of two rings each containing 17 subunits. The packing of the disks in the crystal has been determined, and consists of an approximately body-centred array of stacks comprising two disks each.     

Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus     

Direct visualization of the RNA location in cucumber green mottle mosaic virus and tobacco mosaic virus protein disk

The location of RNA in cucumber green mottle mosaic virus and tobacco mosaic virus protein disks was visualized by a negative staining method as a narrow ring localized at a radius of 4 nm, which corresponds to the location of RNA obtained by X-ray diffraction studies of tobacco mosaic virus. The same ring-shaped stains were observed in the end views of helical rods prepared in acidic solutions from viral protein without RNA. Since such a ring-shaped image could not be observed in end views of natural particles and reconstituted particles composed of protein and RNA, the narrow ring was concluded to indicate the RNA location on the basis of X-ray analysis.     

The tobacco mosaic virus particle: structure and assembly

A short account is given of the physical and chemical studies that have led to an understanding of the structure of the tobacco mosaic virus particle and how it is assembled from its constituent coat protein and RNA. The assembly is a much more complex process than might have been expected from the simplicity of the helical design of the particle. The protein forms an obligatory intermediate (a cylindrical disk composed of two layers of protein units), which recognizes a specific RNA hairpin sequence. This extraordinary mechanism simultaneously fulfils the physical requirement for nucleating the growth of the helical particle and the biological requirement for specific recognition of the viral DNA.     

Electron microscopy of the stacked disk aggregate of tobacco mosaic virus protein. I. Three-dimensional image reconstruction

The three-dimensional structure of the stacked disk aggregate of tobacco mosaic virus protein has been determined from “phase plate” electron micrographs to an effective resolution of about 12 Å. It is a long rod comprised of paired rings of protein (disks), the subunits of which have different conformations according to which ring they belong. The two subunit conformations are such that the rings come close together within a disk near the outer surface of the particle, but between disks on the inside. This property, interpreted on the basis of a polar packing of the subunits, was established from an earlier, lower resolution, study by Finch &; Klug (1971). The present study shows, in addition, that the pairing is contributed mainly by axial distortions of the subunits in one of the rings, the axial distortions of the subunits in the other being largely replaced at lower radii by a tilt or twist and, at higher radii, by a slew. The subunits in the latter ring appear to have a conformation similar to that of the protein molecules in the virus.     

Assembly mechanism of tobacco mosaic virus particle from its ribonucleic acid and protein

Summary The reconstitution process of an infectious tobacco mosaic virus particle from its RNA and protein consists of two steps, formation of the initial complex and growth of the helical rod, the former is the rate limiting step. The protein aggregate, having about 20–30 S, is needed for the formation of the initial complex with 5-end of tobacco mosaic virus RNA. The elongation reaction from the initial complex proceeds even under conditions where both the reconstitution reaction and the formation of 20–30 S protein aggregates do not take place. This indicates that the growth of the helical rod proceeds by stepwise additions of protein subunits or 4 S aggregates. A possible model for assembly process of tobacco mosaic virus particle is presented.     

Circular dichroism and sedimentation studies on the reconstitution of tobacco mosaic virus

Reconstitution of tobacco mosaic virus from its constituents, the coat protein and RNA, was investigated by means of ultracentrifugation and circular dichroism measurement. Tobacco mosaic virus protein forms a 20S double-layer disc under conditions favorable for tobacco mosaic virus reconstitution. Dibromination of the tyrosine 139 residue of tobacco mosaic virus protein prevents formation of the 20S disc.Acidification of the tobacco mosaic virus protein solution causes 20S discs to polymerize into long helical rods. Changes in the CD spectra of tobacco mosaic virus protein in the near-ultraviolet region suggest that stacking of the aromatic sidechains of amino acid residues stabilizes the helical rod. The dibrominated tobacco mosaic virus protein also has the ability of rod elongation under acidic condition. CD studies reveal that assembly of tobacco mosaic virus particles from its constituents is stabilized by the stacking effect between the base residues of RNA and the aromatic residues of tobacco mosaic virus protein.Cucumber green mottle mosaic virus protein, which acts as a substituent for tobacco mosaic virus protein in tobacco mosaic virus reconstitution, was also investigated.     

Structural repeats and evolution of tobacco mosaic virus coat protein and RNA

The three-dimensional structure of the tobacco mosaic virus (TMV) coat protein disk suggests a possible pathway for the early evolution of the virus self-assembly mechanism.The coat protein contains a 2-fold repeated structural pattern in the folding of both its four alpha helices (A,B,C,D), which run alternately forward and back along the radius of the disk, and the four-stranded antiparallel pleated sheet which links these helices to the hydrophobic girdle at the outer rim of the disk. Helices A and B can be approximately superposed on C and D by a screw rotation about a molecular pseudo-dyad axis which lies nearly parallel to the plane of the protein disk. This operation relates 29 pairs of α-carbon positions with a root-mean-square deviation of 1.77 Å. A second pseudo-dyad in the pleated-sheet region relates 14 more atom pairs with a deviation of 2.32 Å and forms a distorted continuation of the relationship between the helices. The helix dyad also relates repeated pairs of functionally important amino acids which take part in intersubunit contacts.We have analysed these structural repeats and tested their significance by comparing them with repeats in other “helix quartet” proteins, cytochrome b⁵ and the hemerythrins, as well as with an irregular helix cluster in thermolysin. TMV is noticeably more repetitive than the others, including hemerythrin which is thought to have evolved by gene duplication.We propose that the primitive TMV coat protein was a dimeric structure of two smaller units paired about a 2-fold axis. Each unit was a pair of helices, linked at the inner radius of the virus rod by a short bend, where the RNA binding site formed, and connected at the outer radius by two short strands of beta sheet. A tandem gene duplication joined the two units and formed the present helix quartet. The flexible loop which now runs into the centre of the virus and connects helix C to helix D developed later. The assembly origin RNA may have evolved from part of the coat protein RNA which codes for this loop.