Tobacco mosaic virus (TMV) was the first virus to be recognized as a disease entity different from bacteria when Beijerinck (1) concluded that it was a new type of infectious agent, "Contagium vivum fluidum." Since then, it has spurred the development of various concepts of viruses, being, among other aspects, the first virus to be purified and crystallized and shown to be composed of protein and RNA (2). Details of the history of TMV are given in Fraenkel-Conrat (3).
TMV is found worldwide and locally can cause an important disease in tobacco. It has a large host range, infecting at least 200 species from 30 families (4). The virus occurs in very high concentrations in infected leaves (> 106 particles per cell) and is very stable, even retaining its infectivity in nonsterile extracts at room temperature for more than 50 years (5). These properties make it very readily transmitted by contact between plants. Man is the main vector, either directly by handling infected and then healthy plants or indirectly by farm machinery. The virus survives in dead crop debris and thus can be transmitted to the next season’s crop at planting time.
TMV is the type member of the tobamovirus genus, which contains 13 species and two other possible members.
1. Virus Structure
The virions of TMV are rod-shaped, 300 nm long, and 19 nm in diameter, and composed of a single-coat protein species encapsidating a single (+)-strand linear RNA molecule of about 6.4 kb. The detailed structures of the virions and coat protein subunits have been determined by electron microscopy and X-ray crystallography. In a virion, approximately 2100 subunits are closely packed in a single right-handed helix of pitch 2.3 nm and with 16 coat protein molecules per turn (Fig. 1). The RNA binds at a radius of about 4 nm, with three nucleotides per protein subunit. There is a central canal of about 2-nm radius. The length of the rod is determined by the length of the RNA, which is fully encapsidated.
Figure 1. The structure of part of TMV. The portion indicated includes 18 turns of the helical array, with 16 subunits/turn.The entire virus consists of about 128 turns.
Much is known about the detailed structure of the protein subunits and their interactions in forming the virion (6). The protein monomer aggregates in solution in various ways, depending on ionic strength, pH, and temperature (7, 8). The structure of one of these forms, a double disk of 17 subunits per disk that is an intermediate in virus assembly, has been determined by X-ray crystallography to 2.8 A resolution. Up to a radius of 4 nm, no structure is resolved, which suggests a disordered state. Much of the rest of each protein subunit is made up of four a-helices, with their distal ends bound together by regions of b-sheet. Both the N- and C- termini of the polypeptide chain occur at the circumference of the disk. The subunits have polar and hydrophobic interactions with each other. In the virion, there are also electrostatic interactions, between both the RNA and protein one involving pairs of carboxyl groups with anomalouspKa values (near pH 7.0) on adjacent subunits. It is thought that these carboxyl groups play an important role in particle assembly and disassembly.
The self-assembly of TMV coat protein subunits has been studied extensively in vitro by the addition of virion RNA to coat protein preparations, which leads to the reassembly of virus particles. Assembly starts at a specific site on the RNA, termed the origin of assembly, which is a region of extensive stem-loop structures about 1100 to 900 nucleotides from the 3′ end. In the initial initiation event, the hairpin loop of the origin of assembly interacts with a double disk of coat protein subunits so that both ends of the RNA protrude from the same side of the disk. The hairpin loop opens up as the RNA intercalates between the two layers of the double disk, which changes to a helical (double-lockwasher) form. A second double disk adds to the first on the side away from the protruding RNA tails and, as it switches to a helical form, it interacts with the RNA 5′ to the origin of assembly. The helical rod continues to grow in the 5′ direction by the further addition of double disks, effectively pulling the RNA through the axial hole until all the RNA 5′ of the origin of assembly is encapsidated. The assembly in the 3′ direction is much slower than that in the 5′ direction and occurs by the addition of small aggregates of coat protein (A protein form) to the helical rod.
The in vivo disassembly of TMV involving the cotranslational disassembly mechanism is discussed under Virus infection, plant.
2. Virus Genome
The genome of TMV comprises 6395 nucleotides and codes for four, or possibly five, proteins (Fig. 2). The first open reading frame, encoding a protein of 126 kDa, ends in an amber stop codon, but this is occasionally read through to also give a protein of 183 kDa. These two proteins are involved in TMV replication and make up the viral RNA-dependent RNA polymerase of the "Sindbis virus supergroup." There are amino acid sequence motifs suggestive of a methyl transferase and helicase domain in the 126-kDa protein and of an RNA polymerase in the readthrough portion of the 183-kDa protein. There is a suggestion that the 54-kDa readthrough part of this protein might also be expressed independently from a subgenomic RNA. The other two downstream proteins, one at 30 kDa and the other at 17.6 kDa, are expressed from subgenomic messenger RNAs. The 30-kDa protein is involved in cell-to-cell movement (see Virus Infection, Plant), and the 17.6-kDa protein is the viral coat protein.
Figure 2. Genome organization of TMV. The upper line represents the single-stranded RNA genome; the circle at the 5′ end indicates the cap, and the tRNA-like structure at the 3′ end is also illustrated. The other two lines are the subgenomic RNAs. The open reading frames are shown by the boxes, and their position on the diagram indicates from which RNA they are expressed. The proteins are distinguished by their approximate molecular weights, in kDa. The stippled box (PI83) indicates the RNA polymerase, that striped (P30) is the protein believed to be involved in movement, whereas that cross-hatched (PI7) is the coat protein; Vindicates readthrough.
The viral RNA has a methyl guanosine cap and leader sequence of 67 nucleotides that is very AU-rich. This leader sequence, termed the W sequence, appears to have no secondary structure and enhances translation (9). The 3′-end folds into a transfer RNA-like structure and accepts histidine.
