Tobacco Mosaic Virus:
It is the first virus discovered to contain RNA as the genetic material. Dimitry Losifovich Ivanovsky (1892) a Russian scientist (Microbiologist), was the first to discover the virus as the causative agent in tobacco plants. Wendell Stanley (1948) was the first to crystallize the viruses, which showed infectivity properties. Viruses can exist as living entities in living cells and also exist as crystal like particles outside living cells. It means they are in threshold of living and non living objects.
Crystal of TMV; http://rybicki.wordpress.com/
Diseased tobacco leaves
Diseased tomato leaves
TMV rod shaped structure; http://www.virology.wisc.edu/
The figure shows one end view of the rod but helical structure of TMV;
The virus appears like a hallow cylinder of 300nm long and 18 nm thick. Mol.wt is 40 million KD mass. It is built up of 2130 similar subunits strung around axial but helically oriented RNA from inside, in circular fashion.
Each of the subunits is 158 amino acids long and 17.5 KD mass and consists of four large a helices, one small helix and 6 b sheets organized into a flat disc with N- and C- terminals found at the periphery. Nearly 16 and half subunits form one circular disc or organize into one turn, with 40 A ^o hallow and 140 A^o thick wall. On the inner surface of the disc RNA is bound to each subunit in each disc. The genomic RNA using its GAA sequences bind to Arginine residues found at 90 and 92 nd position of the subunit protein. In all 129 discs are laid one above the other and they are aligned conformationally like a spiral string of subunits, where 6395 ntds long RNA is bound to each subunit from inside via 3 ntds (GAA). Each of the subunits is cross-linked to one another by salt bridges. At one end of the hollow rod is 3 end of the RNA and at the other end of the rod the 5 end of the RNA is found.
· Length of the rod is 300nm,
· Width of the rod is 18nm,
· Thickness of the wall is 14 nm,
· Hollow of the cylinder is 4 nm,
· Capsids are organized into two layered cylindrical discs,
· Each disc is made up of sixteen and half subunits,
· Each subunit is made up of 158 aa, 17.5 kd each subunit.
· Helix pitch is 23 A^o rise,
· Each subunit protein consists of 4 large and one small a helices and 6 b sheets, with N and C- terminals towards periphery,
· The sequence GAA of the RNA binds to Arginine residues found at 90 and 92-nd position of the subunit.
· Total number of subunits found in each TMV rod like particle is 2130, bound from inside to 6395 nucleotide long RNA, with one 5 end and the other 3end.
· The rod is stable at low pH (5.2), and discs grow well at low pH. At higher pH the discs are unstable and fall apart.
· The genomic RNA is 6395 ntds long and it has positive sense, which means it can be translated as mRNA.
· Under In vitro condition the assembly of the particles into a viral structure takes about 6 hrs, but under in vivo conditions it hardly takes 10 minutes.
The above picture shows the helical organization of capsomeres around the positive sense genomic RNA from inside; http://www.turbosquid.com/
Assembly in the cytosol takes place in sequence; first two layered discs form. At cellular pH 7, nucleation of discs starts on an 18 base pair long helix organized at 1000 ntds from 3 end of the RNA. The secondary structure, with base sequence of 5 AGA AGA AGU UGU UGA UGA 3 of 18 ntds long contains G at every three bases, surprisingly nowhere C ntds are found in the region. The RNA with the said secondary structure first binds to one of the discs from inside, then the second disc is laid from the top and the RNA is drawn into it for binding, thus the discs are laid one above the other till the 5 end of the RNA is completely pulled upwards into the rod. The 3 end of the RNA is still
The diagram depicts how the capsomeres assemble into discs and how the discs get associated with the looped but coiled RNA; http://webappsr2.swau.edu/
hanging loose. During rod building process, the GAA sequence of the RNA binds to each of the discs to Arginine at 90 and 92 nd position. Once the 5 end assembly is completed, assembly of discs at lower end starts growing towards 3 end of the RNA disc by disc. This type of assembly is highly favored at low pH, at which the rod like structure can be organized even without the presence of RNA. These discs as they organize or laid one above the other at the upper end (towards 5 end) or one below the other at the lower end (towards the 3 end), they conformationally change and slide to form one continuous strand in helical orientation.
· The genomic RNA is 6395 ntds long consists of cap at 5 end, which is a distinguishing feature of eukaryotic mRNAs, and it has a tRNA like secondary structure at the 3 end.
· The genomic RNA has positive sense; it is encoded with information to produce three polypeptides, one 126.183kDa, the second with 29.978 kDa and the third with 17.60 kDa proteins.
· The first one, which is not fully characterized, is RNA dependent RNA polymerase, acts on both positive and negative strands. The second, a 29kd product acts as a transporter protein, which facilitates the movement of viral particles across the cells through plasmodesmota. The third 17.6 KD is the capsid protein, which is located at the 3 end of the genome.
· Entry of the virus is through wounds, and once in the cell, it is decoated.
Figure. Current Molecular Biological Conception of TMV, as Represented by a Schematic Diagram of its Genome Organization.
Genome with protein coding regions; http://users.aber.ac.uk/
The 5′‐leader sequence (called Ω) of tobacco mosaic virus (TMV) functionsas a translational enhancer in plants. A poly(CAA) region within Ω is responsible for the translation enhancement and serves as a binding site for the heat shock protein, HSP101, which is required for the translational enhancement. the functional interaction between Ω and other RNA elements known to participate in the recruitment of eIF4G, The use of a fractionated translation lysate revealed that of the two eIF4F proteins present in plants, eIF4F was specifically required for the activity of Ω. The data suggest that Ω is functionally similar to a 5′‐cap and a poly(A) tail in that it serves to recruit eIF4F in order to enhance translation from an mRNA. By Daniel R. Gallie*
· The genomic RNA has all the characteristic features of an mRNA, so its 5 associates with cytosolic ribosomes and translates. Initially translation appears to terminate at the end of the first reading frame and the product is RNA dependent RNA polymerase, looks like a monomer.
· The polymerase enzyme recognizes the tRNA like structural feature at the 3 end of the genome and uses it as the primer for .yet it is not clear how the replication initiation takes place on the plus strand to produce negative strand.
· Using tRNA like or tRNAs for replication is not an unique feature, for this feature is exploited by reverse transcriptases during retro viral genome replication.
· Once the full length (-) RNA is produced, it is again used by the same RNA polymerase or Replicase to produce the positive strand. In the beginning the products are sub-genomic (+) ve RNAs, in the sense only one reading frame length of the RNA is transcribed, no full length (+) RNA.
· Most abundantly produced RNA is capsid mRNA for 17.6kd protein. The second most mRNA produced is of transport protein. The third mRNAs is for enzyme proteins.
The RNA is first translated and the replication enzymes produced use the positive RNA strand for the synthesis of negative strand and then the negative strand is used for the production of positive strand, which associates with capsid discs for the assembly of TMV particles; https://www.apsnet.org
· When capsid proteins are produced to an optimal level, the enzyme starts producing, full length (+) genomic RNA. How this switch from sub-genomic RNAs to full length genomic RNA synthesis takes place is not fully understood even after 80 years of its discovery i.e. in 1920s. Another way to explain the production full length (+) RNA synthesis and production of proteins, is explained, as the (-) RNA strand is used directly for the production of a full length (+) RNA, and it is then translated differentially, to synthesize large number of capsid proteins required for viral production, strangely this part of the RNA is found at 3 end of the RNA.
The above diagram shows how the TMV particles form complexes and bind to cell walls and enter through plasmodesmota into cells; http://www.mindfully.org/
Cell to cell transmission is through Plasmodesmata; http://www.mcb.uct.ac.za/
The TMV virus, though detected in Tobacco plants, first, causing yellow mosaic virus has been known to infect other plants especially Solanaceae members including tomato plants. As this disease can spread fast it can devastate a large area of crop fields. The other important crop plant that is seriously affected by this virus is tomato, on which it causes Tomato Mosaic disease (ToMV disease); combined with tomato leaf curl virus affect 60 to 80 percentage of tomato crop and it is deadly in India).
Disease resistance to these viruses has been developed based on capsid mediated resistance and RNA mediated resistance. RNA mediated resistance is by ds RNA mediated mi/siRNA mode, another is ds mediated PKR mode and RNase L mode, and developing Transgenic tomato plants is in progress (grk.raj in IAHS, Bangalore).