# Tobacco Mosaic Virus - Gülbeyaz Aygün(1605A023)

# ****Tobacco Mosaic Virus(TMV)****

Tobacco Mosaic disease is caused by Tobacco Mosaic Virus (TMV). This virus has worldwide distribution and primarily infects tobacco and tomato, but more than 350 species are susceptible. The different characteristics of this plant virus are as follows:

1. TMV is a rod-shaped particle which is 300 nm long 15-18 nm in diameter(Figure 1). It possesses SSRNA and a protein coat.

Figure 1: Electron microscopy picture of tobacco mosaic virus(TMV)
Image source : P.Saravanan,"Tobacco Mosaic Virus" *Virology*, Plant Virology, Prototype Viruses, 2017, pp.135.

2. TMV is difficult to inactivate, and can survive for almost 5 years in dead, dried tissues and for many months in living plant tissues.
3. There are many strains of the virus that vary in their virulence causing severe to mild symptoms. Virus is spread from plant to plant through injuries caused by crop worker, contaminated equipment and chewing insects.
4. These viruses are found in dead plant tissues and debris, on contaminated equipment, in contaminated soil, greenhouse containers, bedding, tools, and in living hosts, including weeds like horsenettle, Solanum carolinense, and other crop plants (tomato, pepper, and eggplant).

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***Symptoms of the TMV disease are:***
* a. Tobacco leaves become mottled with light and dark green areas; leaves become distorted; puckering or blistering takes place especially in areas of new growth.
* b. In tomato, mottling of leaves occurs and leaflets become long and pointed and there is stunting of plant growth.


Image source: [researchgate](https:https://www.researchgate.net/publication/51478099_Detection_of_Tobacco_mosaic_virus_and_Tomato_mosaic_virus_in_pepper_and_tomato_by_multiplex_RT-PCR/figures?lo=1//)

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# Structure and Self-assembly

The stability of the TMV particle accounts for its having been the first virus to be identified, purified to homogeneity, and then biochemically and biologically characterized. The characteristic features of the physical structure includes:

1. This rod shaped virus is of length 300 nm and radius 9 nm, with a central hole of radius ~ 2 nm.

2. The subunits are arranged in a single helix which is right handed with 16% protein subunits per turn.
3. The ssRNA binds at a radius of ~4 nm with 3 nt per protein subunit and the length were either 6395 or 6398 nt (due to polymorphism near 5' terminus), this corresponds to ~2140 protein subunits and 130 turns of the viral helix

[Figure 3: Structure of TMV(simple)](https://en.wikipedia.org/wiki/File:TMV_structure_simple.png)

4. The main form of protein aggregates at high pH and low ionic strength is a mixture of monomer and small aggregates, collectively known as "A-protein". The two-layered disk of TMV protein is central among the aggregates.
5. The main form of protein aggregates at high pH and low ionic strength is a mixture of monomer and small aggregates, collectively known as "A-protein". The two-layered disk of TMV protein is central among the aggregates.
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# Self Assembly
Self assembly involves two major steps:
* Nucleation
* Elongation

**Nucleation** involves stacking of subunits along the RNA strand and is highly selective for the TMV RNA species (very high specificity).
**Elongation** or **growth** is bidirectional as observed in the Vulgare strain of TMV, but is faster in the 5'-direction and is ~5-fold. Aggregates of 33 protein molecules form the double disk. This combines with viral RNA.
Attachment of the nucleic acid to the protein aggregate begins at a unique region on the viral RNA called as origin of assembly site (OAS) about 900 nucleotides from the 3'terminus of TMV RNA.
Co-translation disassembly is defined as the process in which the protein coat is displaced at the 5’ end by ribosomes in the host cell.

Figure 4 : Schematic representation of Selfassembly of Tobacco Mosaic Virus
Image source : [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1692540/pdf/10212933.pdf]

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# Infectivity
Viral ingress into the plant is by a small, healable wound. There are no known vectors observed. The virus movement from cell to cell occurs through plasmodesmata mediated by virus coded 30 kDa protein. The long distance virus movement in the host at a fast rate is through stem phloem tissue without any replication. The symptom determinants include systemic mosaic and hypersensitive necrotic local lesions, which are determined by N-gene of the tobacco plant.

[Figure 5: Infectivity of TMV to plant leaves](http://www.plantphysiol.org/content/153/4/1859)

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# Replication

***Gene organization***

TMV RNA itself is an mRNA of 6395 nt, referred to as (+)-sense RNA, containing 5 genes. The 5' proximal gene encodes two co-initiation proteins of 126 kDa and 183 kDa. The 3 genes located internally in the genome are not expressed from genomic RNA but rather from subgenomic (sgRNA) RNA generated during the course of infection.
SgRNAs include I, mRNA of 54 kDa (begins at 3405, 2991 residues), I, mRNA of 30 kDa (begins at 4838, 2058 residues) and LMC-RNA of 17.5 kDa (begins at 5703, 693 residues).
Both genomic RNA and LMC-RNA have been shown to be capped at 5' end by m 7G. The 3'-end of TMV RNA ends with the sequence -C-C-C-A and can be charged with an amino acid (histidine). This region is non-coding and can be folded into a tRNA-like structure preceded by a series of four pseudoknots.

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# Life Cycle of TMV

Virus enters by force, and uncoating and exposure of 5'ORF of TMV RNA is facilitated by Ca2+ binding sites (~10–7 M intracellular Ca2+).

Uncoating is a bi-directional process using co-translational mechanism for the 5' → 3' direction.

Disassembly and replication are coupled events. Three virus encoded enzyme activity seen in replication of TMV RNA viz., RNA-dependent RNA polymerase (RDRP) that synthesizes (-)-RNA using an RNA template, a helicase involved in RNA displacing activity, and methyl transferase leads to 5' capping of RNA.

In protoplast (-)-strand synthesis ceases 6-8 h post inoculation, but (+)-strand synthesis continues for a further 10 h which are encapsidated into virions.

5'ORF of TMV encode 126 kDa protein, the stop codon of which is read through to give a 183 kDa protein.

**Figure 6: Tobacco Mosaic Virus life cycle**
Image source: P.Saravanan,"Tobacco Mosaic Virus" *Virology*, Plant Virology, Prototype Viruses, 2017, pp.141.

Both of these proteins have helicase motifs and 183 kDa in addition has RDRP motif. Viral RNA binds a replicon (formed by combination of 126 K and 183 K proteins-active replicase) molecule at its 3'-end and become a template for (-)-strand synthesis. This structure is called “Replication form (RF)".

3'-UTR of TMV RNA can be folded into three structure domains; a 3'-domain mimicking a tRNA acceptor branch, an analogue of a tRNA anticodon and an upstream domain comprising 3 pseudoknots each containing 2 double helical segments.

30 kDa protein of TMV was identified as MP, which has the abilities to bind nucleic acid, to increase plasmodesmatal SELs, to facilitate movement of RNA to neighbouring cells as microinjection, and interacts with cytoskeletal elements (microtubules in the protoplasts and pectin methyltransferase (PME) of cell walls) of the cell.

Rate of movement from upper to lower epidermis was found to be 8 um/h.

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