VIRAL STRUCTURES

The morphological description of virus began in 1935. Stanley applied methods of protein fractionation to the sap of diseased plants and purified particles of tobacco mosaic virus in the form of liquid crystals. The fine paracrystalline needles obtained even when highly diluted proved capable of reproducing the disease and Stanley concluded that they were “autocatalytic protein” that needed living cells in order to multiply. In 1936 a first bacterial virus was described as a nucleoprotein containing DNA. The study and description of these infectious molecules capable of organizing the multiplication of their nucleic acid and then transferring it to from one cell to another then began.

Architecture of the virion

The viral particle, also called virion, is composed of a nucleic acid and a proteic shell called a capsid, depending upon the virus, it may also have an outer coat derived from membranes of the host, enzymes, and zinc or calcium ions in small quantity. The capsid surrounds the nucleic acid; it is composed of subunits. Each subunit is a polypeptide chain of around 150-400 amino acids, coded by the viral nucleic acid; it is one of the viral proteins in which the sequence is most variable among species, strains, and isolates. On the outside of the cell, the capsid protects the nucleic acid. Inside the cell, it plays a particularly important role in various processes, depending on the virus.

The structure of the capsidial subunit and its relationship with viral RNA

When the sequence of a polypeptide chain is established, the stable domains of the structure resulting from its folding can be predicted: α-helices generated by the rotation of the polypeptide chain on itself, β-sheets in which the chain folds on itself and turns and loops that connect the sheet and helices into a globular mass. These predictions of the secondary structure are not entirely reliable, and they must be confirmed by referring to proteins already known by means of crystallographic methods. The tertiary structure of a polypeptide chain results essentially from the spatial organization of helices and sheets; it is stabilized by non-covalent interactions between amino acids: hydrogen bridges and saline bonds between the polar groups, hydrophobic interactions between non-polar groups. These forces act within subunits as well as between subunit.

The subunit of the TMV capsid has a high proportion of secondary structures: 50% in α-helices, 10% in β-sheets as well as loops. Four antiparallel helices (denominated LS, RS and LR,RR)form the basis of the structure. Nearer the outside, two short helices are close to the NH₂and COOH ends, which are found on the outer surface. On the lateral faces, there is an alternation of hydrophilic and hydrophobic zones.

Interactions between the subunits

Interactions between the subunits stabilize the viral particle either through lateral bonds between subunits contiguous on the helix or between superimposed units on two successive turns.

1.     The lateral bonds are essentially non-polar. They are realised by aromatic and aliphatic amino acids that form a hydrophobic bond close to the outer surface; two other highly hydrophobic regions are located towards the inside of the particle.

2.     The bonds between two superimposed subunits are mostly electrostatic. A large zone of non- specific interactions is found towards the inside of the helix, a second less extensive zone is located at a radius of around 5.5nm. A third zone located towards the outside of the helix stabilizes the subunit.

Protein-RNA interactions. Viral RNA is associated at each subunit by the intermediary of the three bases, in two different ways:

1.     The bases are linked to amino acids of the LR helix of the subunit located below by very close interaction between the phosphates of the negatively charged RNA and the basic arginines 90 and 92, but there are other bonds between each of the bases and several amino acids.

2.     Other electrostatic interactions occur between RNA and proteins, especially with amino acids of the RR-segment of the subunit located below.

The interactions between RNA and proteins is ultimately realized by three types of the bonds:

a)     Electrosatic forces between arginines and phosphate groups of the RNA.

b)     Specific hydrogen bonds, including with the group 2’-hydroxyl of ribose.

c)     Non–specific hydrophobic interactions with the LR helix, which allows encapsidation of the complete sequence. Several amino acids implicated directly in the RNA bonding are conserved between strains.

Despite the stability of the TMV virion, the RNA must be able to decapsidate and encapsidate

The structure of the particle must ensure protection of the RNA as well as permits its disassembly during infection; these two states are possible because of a metastable equilibrium whose energy is close to the minimal energy state. Apart from the forces that stabilize the particle, there are repulsive electrostatic forces between the TMV subunits, due to carboxyl groups that are neutralized in the particle by protons or calcium ions. During entry into the cell, the change in pH and calcium concentration allows repulsive forces to destabilize subunits of the last turns of the helix. Starting at the 5΄ end of the viral RNA, the process of decapsidation is linked with the attachment of ribosomes to RNA (Lu et al. 1998). Decapsidation proceeds from the 5΄ end to 3΄ end with the ribosomal complex and very quickly covers two-third of the RNA, i.e the end of the replication genes. In a second step, decapsidation proceeds inversely form 3΄ end to 5΄ end and terminates in the region that contains the origin of assembly. The newly synthesized replication proteins participate in this second step (Wu and Shaw 1997). Decapsidation is thus an active phenomenon, linked to translation and replication.

If the TMV particle can dissociate into subunits and RNA in vitro, it can also reassemble by simply mixing the RNA and subunits and by adjusting the pH, ionic forces, and concentrations (Fraenkel-Conrat and Williams 1995). These assembly experiments have shown that information necessary for encapsidation, a highly specific phenomenon, is contained in the components of the structure. The starting point of encapsidation is the formation of polarized double disc (2 x 17 subunits) that recognizes the encapsidation origin of the viral RNA, as structure in three successive hairpins located in the gene of the 30 kDa protein about 900 residues from the 3΄ end (Jonard et al. 1977). This secondary structure and a sequence with repeated AAG motifs (including guanine 122, which is at the beginning of the process)are recognized by one or two subunits of the double disc, which leads it to its development into a helical structure. At this stage, the 3΄ and 5΄ ends of the RNA are found on the same side of the growing rod. The elongation occurs by the positioning of the subunits next to each other, chiefly on the 5΄ end, i.e, on the side of an RNA loop that slides to the extent that the RNA chain gets established between the subunits, without specificity of sequence. Encapsidation is complete in the same fashion on the side on the 3΄ end. The hydrogen bonds that are established between the subunits make it a highly cooperative process (Namba et al. 1989). In another rod shaped virus, PCV, two different sequences derive the encapsidation of RNA -1 and two other unrelated sequences the encapsidation of the RNA-2 (Hemmer et al. 2003).

Through experiments in assembling viral particles by mixing RNA and capsidial subunit of the two strains of TMV, Fraenkel-Conrat and Singer established in 1957 that the viral RNA codes for its capsid protein, thus demonstrating for the first time that RNA can carry genetic information.