The following points highlight the three major sub-viral entities. The sub-viral entities are: 1. Viroids 2. Virusoids 3. Prions.
Sub-Viral Entity # 1. Viroids:
Viroids are a novel class of sub-viral pathogens that are found to cause diseases on plants and are the smallest known infectious agents.
They are also known by the names ‘metaviruses’ or ‘pathogene’ and differ basically from viruses in at least following features:
(i) Virus-RNA is enclosed in a protein coat while the viroids lack any protein coat and apparently exist as free-RNA,
(ii) Viroid-RNA is of small size consisting of 246-375 nucleotides as compared to 4-20 kb of virus-RNA, and
(iii) Viroid-RNA consists of only one molecular species only, while many virus-RNA exist as more than one molecular species within the same capsid.
The first viroid was discovered by T.O. Diener in 1971 who found it to be the causative agent of Potato spindle tuber disease (Diener, 1979), the disease previously considered to be caused by Potato spindle tuber virus.
Since then, several other plant diseases are now known to be caused by viroids; some important ones are Chrysanthemum chlorotic mottle disease, Chrysanthemum stunt disease, Citurs excortis disease,Coconut cadang-cadang disease, Tomato bunchy top disease, Tomato apical stunt disease etc.
Viroids are small, circular, single-stranded RNA molecules ranging from 246 nucleotides (Coconut cadang-cadang viroid) to 375 nucleotides (Citrus excortis viroid) in size. Their molecular weight is low and ranges from 85,000 to 1,30,000 daltons. The extracellular form of viroid is naked-RNA, there is no oapsid of any kind.
Even more interestingly, the RNA molecule contains no protein encoding genes and, therefore, the viroid is totally dependent on host function for its replication. Although the viroid is a single- stranded circular RNA molecule, there is such considerable secondary structure possible that it resembles a short-stranded molecule with close ends (Fig. 11.10).
Viroids seem to be associated with the cell nuclei, particularly the chromatin, and possibly with the endomembrane system of the host cell. There is evidence that viroids replicate by direct RNA copying in which all components required for viroid-replication including the RNA polymerase are provided by the host.
Branch et al. (1981), Owens and Diener (1982) and Branch and Robertson (1983) have proposed the following mode for viroid (Potato Spindle Tuber Viroid; PSTV) replication (Fig. 11.11).
The infecting viroid strand (marked ‘+’) enters a cell, moves into the nucleus and initiates the synthesis of minus (-) strand (i.e., the complementary strand) by a rolling circle mechanism proposed earlier by Brown and Martin (1965) for replication of certain viral RNAs.
The linear (-) strand of RNA then serves as a template (complementary) for replication of strand of (+) RNA. The (+) RNA is subsequently cleaved by enzymes that release linear, unit length viroid (+) RNAs, and these circularize and produce many copies of the original viroid RNA.
Viroids possibly cannot be transmitted as naked RNAs because of their susceptibility to nuclease enzyme. They, however, are protected from this enzyme-attack by being localized within the nuclei of infected cells (Sanger, 1979). Presumably, the viroids are transmitted in association with pieces of nuclei or chromatin and not as free RNA.
Their transmission from diseased to healthy plants takes place primarily by mechanical means, i.e., through sap carried on hands or tools during propagation or cultural practices, and by vegetative propagation. No specific insect or other vectors of viroids are known.
Origin of Viroids:
Origin of viroids is still speculative as there is no sufficient knowledge available in this regard.
However, there are different views to explain the origin of viroids, the most favoured views are as follows:
(i) The viroids are considered to have originated by circularization of spliced-out intervening sequences (introns) during RNA splicing. The introns are usually considered as nonsense sequences that possibly rapidly degrade. If these excised sequences (introns) undergo extensive intramolecular base- pairing and become circularized, they may become stabilized, do not undergo degradation and give rise to viroids.
These ‘viroids’ may possess the relevant recognition sites and could be transcribed successfully by RNA dependent RNA polymerase enzyme. Interestingly, introns, including the ones of viroids size, have been observed to undergo circularization.
In the light of this view, the viroids may be considered ‘escaped’ introns and, like self-splicing introns, appear to be the remnants of an ‘RNA World’ evolved during the early stages of biological evolution.
(ii) Watson et al. (1987) emphasize the other view. According to them the viroids are supposed to be the ‘primitive viruses’ and must have originated from cellular RNAs. It has been found that RNA synthesis on RNA template takes place in most of the healthy plants. Viroids would have originated from this RNA as they did not induce the biosynthetic machinery of their host from their own replication.
Sub-Viral Entity # 2. Virusoids:
Similar to viroids, the virusoids are small, low molecular weight, circular RNAs; they are always associated with a larger RNA molecule of a virus. The virusoids were discovered by Randles (1981).
It is thought that some virusoids are necessary for the replication of RNA of the virus with which they are associated, and may form part of the viral genome. One virusoid has been found associated with Velvet tobacco mosaic virus.
Other virusoids have been found to be more like a satellite, i.e., extra RNA associated with virus capable only of replicating in cells infected by the virus. It has also been found that virusoids produce such structures in infected cell suggest that thereby that their replication cycles resemble those of the potato spindle tuber virusoid and other virusoids.
Sub-Viral Entity # 3. Prions (The Puzzling Proteins):
C. Gajdusek (1957) came across a mysterious disease in New Guinea tribals, which was later named as ‘Kuru’, and prepared neuropathological specimens from a person who died of Kuru. Williair Hadlow who was working on scrapic disease of sheeps and goats examined Gajdusek’s neuropathological specimens and observed during 1957 remarkable similarities between the abnormalities found in brains of Kuru victim and the sheeps and goats dying of scrapie.
Similar observations were made by British investigators T. Alpher, D. Haig and M. Clark during 1966. In 1970s S.B. Prusiner, a biochemist at the University of California (USA), with his coworkers, initiated the isolation and identification of the infectious agent of scrapie.
After exhaustive research for a decade, he in 1982 discovered that the disease is caused by a proteinaceous infectious particle which he christened as ‘prions’ (derived from Proteinaceous and Infections). S.B. Prusiner has been awarded Nobel Prize in 1997 for the discovery of prions.
Prions represent the other extreme from viroids. They are considered to be devoid of their own genetic material (DNA or RNA) and consist of just a single, two, three, or more protein molecules i.e. a prion is merely an infectious protein. The discovery of prions has threatened the universally accepted concept that only the genetic material (DNA, in some cases RNA) is infectious.
If prions lack their own nucleic acids and are merely proteins, a very important question requires an answer. How can a protein enter a host cell and direct the process of replication? To answer this question a large number of hypotheses have been put forward.
An interesting hypothesis has been given by a group of scientists from the MRC Neuropathogenesis Unit at Edinburg. This hypothesis states that the existence of small piece of ‘DNA gene’ (also called PrP gene) is necessary to encode the amino acid sequence of prion protein at the time of its replication.
This ‘DNA gene’ is a component of the host genetic material (host DNA). The prion protein presumably serves as a promoter of ‘DNA gene’ expression.
Structure and Chemical Nature:
Prions are 100 times smaller than viruses, contain only protein, are heterogenous in size and density, and can exist in many molecular forms. Gel electrophoresis investigations have revealed that prions possess an apparent molecular weight between 27,000 and 30,000 daltons.
Electron microscopic studies have shown that a large number of prion molecules (~1000) aggregate together to form a composite structure called ‘prion-seed’. The latter are typically 100 to 200 nm in length and 10-20 nm in diameter.
The chemical nature of the prions, as stated earlier, is considered to be proteinaceous and they have no nucleic acids of their own. This has been indicated by the various experimental evidences gathered so far. This aspect of prions has been investigated by treating them with nucleases (the enzymes that digest nucleic acids) and proteases (the enzymes that digest proteins).
It has been observed that the nucleases have no effect on prion infectivity, whereas proteases can drastically reduce a prion infectivity. In addition, prions show high resistance to ionizing and ultraviolet radiations, which act mainly on nucleic acids.
Prion diseases, collectively called as transmissible spongiform encephalopathies (TSEs), are degenerative disorders of the central nervous system (neurodegenerative diseases) leading to motor dis function, dementia, and death. In all disorders now referred to as prion diseases, spongiform degeneration and astrocytic gliosis is found upon microscopic examination of the central nervous system.
Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the cellular prion protein (PrPc), a constituent of normal mammalian cells, into infectious protein (PrPSc). However, some important prion diseases are listed in Table 11.1.
Cellular PrP (PrPc) Conversion into Prion (PrPsc):
The host cell contains PrP gene that encodes PrP (for prion protein). Prions seem to be composed exclusively of a modified isomer of PrP designated PrPSc (Sc = scrapie-associated). No differences in the primary structure of PrPc and PrPSc were detected, suggesting that they differ in their conformation.
The normal, cellular PrP, denoted Prpc (C = cellular) is converted into PrPSc whereby a portion of its α-helical and coil structure is refolded into β-sheet. This structural transition is accompanied by profound alterations in the physicochemical properties of the PrPc. Two models for the conformational conversion of PrPC to PrPSc are proposed.
(i) “refolding” model and (ii) “nucleation” or “seedling” model. The refolding model (Fig. 11.12A) proposes that PrPc unfolds to some extent and refolds under the influence of a PrPSc molecule and that the two states are separated by an activation energy barrier.
The nucleation or seedling model (Fig. 11.12B) postulates that PrPC is in equilibrium with PrPSc (or a precursor thereof), that the equilibrium is largely in favour of PrPC and the PrPSc is only stable when it forms a crystal-like aggregate of PrPSc (sown dark) called multimer (or seed).
Multimer (or seed) formation is rare. However, once a multimer (or seed) becomes present, monomer addition ensures rapidly. These multimers (or seeds) are continuously fragmented generating increasing surfaces for monomer addition.
Prion diseases are so far unique among conformational diseases in that they are transmissible, not only experimentally but also by natural routes. While prion diseases are not contagious (i.e. by direct contact), they are transmitted naturally perorally (predominantly by ingestion) and parenterally.
After oral uptake, the prion penetrates the mucosa through M cells of gastrointestinal tract and enter into the Peyer’s patches as well as the enteric nervous system. Depending on the host, spleen and lymph nodes are sites in which prions replicate and accumulate.
It has been suggested by Huang and co-workers in 2002 that myeloid dendritic cells mediate transport within the lymphoreticular system (LRS). From the LRS and likely from other sites, prions proceed along the peripheral nervous system to finally reach the brain, either directly via the vagus nerve or via spinal cord (Fig. 11.13).