ABSTRACT
VirusesVirion components, including viral genome, CPs forming the coat around the genome, and membranes associated with nucleocapsids (NCs), come together in a process called assembly. The self-assembly process of viruses was first demonstrated by Fraenkel-Conrat and Williams in 1955 [21]. This study showed that disassembled TMV particles could be reconstituted in vitro and the particles maintained their infectivity. Virion assembly occurs basically with two mechanisms: (i) They are coassembled with the genome, or (ii) viral genomes are packaged into an empty preformed procapsid in a process powered by nucleoside triphosphate (NTP) hydrolysis. The assembly process is driven by the laws of thermodynamics, meaning that the subunits forming the particle are energetically in a more favorable environment within the virion than the free subunits [22]. The structure of icosahedral virus particles can be described on the basis of the triangulation number defining the possible icosahedral surface lattice designs, quasi-equivalence describing the nearly equivalent bonding, and self-assembly. As many copies of one protein compose the particle the required information for selfassembly resides in the specific bonding patterns of the individual proteins, although with some viruses the assembly process is regulated by scaffolding proteins that are not present in the final virion [23]. Cementing or glue proteins may exist in final virions to give stability, and some other proteins may be incorporated into virions to serve specific functions like binding of the virus to its receptor on the cell surface. To fully understand the assembly mechanism it is important to know the composition of the virion as well as all intermediates formed during the assembly pathway. The biology of virion assembly as well as the requirements to form stable mature particles set boundaries for the doable genetic and chemical modifications of
the virions. Therefore it is especially important for ENC applications to solve the surface exposed areas that do not take part in CP interactions needed in the assembly pathway or for the stability of the mature virions. 1.2.1 Particle Formation via Coassembly
The capsids of helical viruses, both rod-shaped and flexuous filaments, and small icosahedral RNA viruses are coassembled with the genome. Helical viruses are viruses that have their RNA enclosed in a protective shell consisting of identical protein subunits arranged in a helical manner. This basic structure was first proposed by Crick and Watson in 1956 [4]. Their studies led them to conclude that the structure of small viruses consisted of identical units with specific bonding properties arranged to form a close surface around the viral genetic material, the main events being nucleation and elongation in the order mentioned. Virus assembly and stability of virus particles is governed by several molecular interactions. Viruses of the genera Tymovirus and Comovirus, for example, are stabilized mostly by protein-protein interactions and hence can form VLPs in the absence of viral RNA, while viruses of the genera Alfamovirus, Bromovirus, and Cucumovirus are primarily stabilized by RNA-protein interactions and require other virus-encoded proteins for successful packaging [24, 25]. In spite of these two being the major acting forces, other sequence-dependent/independent RNA-protein interactions as well as structure-dependent interactions are also important. One amongst other constraints in coassembly of icosahedral viruses is the dimension of the capsid, which inflicts a restriction on the size of the RNA to be encapsidated. Studies on turnip crinkle virus (TCV) by Qu and Morris exemplify this aspect [26]. The filamentous virus structure is more flexible in this respect, as the virion size can increase with increasing genome size, as shown with potyviruses [27]. Next we attempt to picturize the assembly process of small simple viruses using TMV as a model to enlighten the major steps. 1.2.1.1 Assembly of TMVTMV is a rod-shaped RNA virus, 300 nm in length, 18 nm in diameter, and a central radius of 2 nm [28]. Its virus particle contains about
2100 protein subunits enclosing a single RNA molecule. Hence, RNA makes only 5% of the particle, while 95% contains the protein shell. The length of the protein shell seems to be dependent on the length of the viral RNA. Intensive and informative studies on the in vitro assembly process enabling the proper understanding and uncovering of the TMV assembly have been communicated. These studies are used here as a prime example of the simple assembly process involving condensation of genomic single-stranded RNA (ssRNA)(+) and CPs. The major component of the TMV rod is the 17 kDa CP comprising 154 amino acids. Depending on certain conditions, pH being the most important of all, protein monomers constituting the virion can self-assemble to form different aggregates (Fig. 1.3).
