MicrobiologyBytes: Virology: Viroids Updated: May 23, 2006 Search

Viroids and Virusoids

Just as nucleic acids can carry out enzymatic reactions, proteins can be genes.   Reed Wickner.

Viroids, virusoids and prions are unusual infectious agents characterised by having a very small genome and in the case of prions, possibly no genome at all.

Viroids are common plant pathogens which are a serious economic problem.
25 different viroid sequences have been determined and numerous variants identified :

Avsunviroidae: e.g. avocado sunblotch viroid, peach latent mosaic viroid
Pospiviroidae: Subgroup 1: potato spindle tuber viroid, coconut cadang cadang viroid, tomato plant macho viroid
Subgroup 2: citrus bent leaf viroid, pear blister canker viroid

 

Pospiviroidae are a large group, only 2 members of the Avsunviroidae are currently known. The RNA genomes of viroids are 246-375 nucleotides in length and share many similarities:

  • They are all single stranded covalent circles
  • There is extensive intramolecular base pairing
  • A DNA-directed RNA polymerase makes both plus and minus strands
  • Replication does not depend on the presence of a helper virus
  • No proteins are encoded

The structure of a Pospiviroid is indicated schematically below:

Viroid genome

The classification described above is based on analysis of the central conserved region (CCR). Members of the Avsunviroid group are clearly different to those described above . They lack a CCR and possess a ribozyme activity (a ribozyme is a catalytic RNA molecule, in this case RNA cleavage is the ribozyme activity). Additionally it is speculated that Avsunviroids may replicate in chloroplasts whereas Pospiviroids replicate in the nucleus and nucleolus. 3 enzymatic activities are required for viroid replication, an RNA polymerase, an RNAse and an RNA ligase.

Avsunviroids probably replicate via a symmetric rolling circle mechanism, whereas Pospiviroids probably use an asymmetric mechanism. By this I mean the +ve infecting circular RNA strand of a viroid serves as a template to make a large linear multimeric -ve strand. RNA pol II is probably the enzyme which does this. Pospiviroids with an asymmetric replication pathway then make +ve RNA from this long linear molecule. A host RNAse activity cleaves the +ve strand into unit viroid lengths. This molecule is then ligated to form a circular viroid. In Avsunviroid replication the long -ve RNA is self cleaved by the associated ribozyme activity. The RNA circularizes to form a -ve circle. A second rolling circle event makes a long linear +ve strand which is again cleaved by the ribozyme activity. The short viroid RNA is then ligated to the circular form.

Viroid replication

 

There thus seems to be fundamental differences between the two groups of viroids, presumably reflecting different origins.

Probably there is more than one mechanism responsible for viroid pathogenesis. Recent evidence suggests that one pathway is due to viroid RNA activating a plant RNA activated protein kinase, or PKR (analogous to the PKR enzyme activated by viral RNAs in mammalian cells). Protein synthesis is reduced and this causes pathogenic effects. In the case of potato spindle tuber viroid, there is a good correlation between a strains pathogenicity and its ability to activate PKR in vitro.

Origins and spread A number of features of viroids suggest they may have originated in the hypothetical prebiotic RNA world. They can possess a ribozyme activity.They are GC rich which would attenuatethe low fidelities of replication activities. They are circular and so do not require start and stop functions for replication. They move within a plant, probably in association with host proteins via the phloem vascular channels and plasmodesmata cell contact points. They seem to form a quasi-species population and can recombine. Spread, at least in commercial crops, seems to be very dependent upon the activities of Man. Viroids of a particular type are widespread in some areas and absent in others.

Tabler M, Tsagris M. (2004) Viroids: petite RNA pathogens with distinguished talents. Trends Plant Sci. 9: 339-348.

 

Virusoids and Satellites

Five virusoid RNA genomes are 220-338 nucleotides long, circular, single stranded and possess a ribozyme activity. They can replicate in the cytoplasm using an RNA-dependent RNA polymerase. This enzymatic activity is common in plants but not found in animal cells. They depend on a helper virus for replication. This helper virus also encapsidates them, e.g:

Subterranean clover mottle virus satellite RNA: Helper - Sobemovirus

Virusoids belong to a larger group of infectious agents called satellite RNAs, found in bacteria, plants, fungi, invertebrates and vertebrates. Satellite genomes encode proteins, satellite viruses encode capsid proteins but are still dependent upon a helper virus for replication. Examples include:

 

HDV

 

They can be spread by vegetative propagation, within seeds or by direct inoculation either by insects or man.

There are similar infectious agents which infect animals, e.g. newt satellite 2 transcript. One such agent infecting humans is the hepatitis delta virus (HDV).
HDV was first identified in the 1970s in Australia as a nuclear antigen, the delta antigen. Subsequently, it was found to be the cause of a particularly virulent form of hepatitis known as type D hepatitis.

Common in indigenous natives of S.America, method of transmission not understood. Can be transmitted perinatally. In the West, transmission is associated with drug abuse and transfusion of blood products. It seems prudent to assume that it can also be transmitted sexually. No specific treatment.
The delta antigen is associated with a defective pathogen which is obligatorily associated with Hepatitis B helper virus:

HDV viroid

This virus has a circular single stranded RNA genome of about 1700 nucleotides. It has a ribozyme (RNA cleavage) activity. This is the smallest known genome for an animal virus.

RNA and delta antigen (195 AA) are packaged in a Hepatitis B particle. No DNA intermediate has been detected during the replication phase and it is thought that replication occurs by RNA directed RNA synthesis using a DNA dependent RNA polymerase. Certain parts of the genome and the pattern of replication is of course similar to a viroid. One difference is that mRNA and a protein - the delta antigen - are made.

The encapsidated RNA is (-)ve sense strand so (+)strand RNA synthesis to make the mRNA is required. These RNAs are nuclear associated and in any case circular RNAs are not good templates for protein synthesis. About 10% of the antigenomic strand is cleaved and polyadenylated and serves as a 800nt mRNA. RNAse activities tend to be exonucleolytic rather than endonucleolytic. The fact that the genomic strands are circular probably contributes to the agents stability.

The different RNAs made by this virus are indicated in the diagram shown below: The genomic strand is 70% Watson Crick base paired and rod like in gross structure.

Viroid genome

The delta antigen is a 22kd nuclear phosphoprotein essential for replication and particle formation. It is basic and associates specifically with the RNA genome thereby stabilizing it. Two forms are made differing by 19 amino acids at the C-terminus. The large form is a dominant inhibitor of genome regulation and directs genome packaging into HBV virus particles. This packaging is due to farnesylation of a Cys residue 4 AA from the C-terminus. Recently a host protein interacting with the delta antigen has been identified. In fact sequence similarities suggest it is a cellular homologue of the delta antigen. This suggests that it may be able to modulate viral replication. It may also suggest that HDV originated from a viroid like element which then "captured" a cellular transcript. Probably the genomic strand transferred onto the mRNA for the cell homologue of the delta antigen. This was copied into the antigenomic strand and stabilize as part of the genome.

These agents all have a small genome. They replicate, spread, and if appropriate, form particles, in much the same way as all of the other viruses you will hear about during this course. One could almost say that there is a continuum between naked replicating transmissible RNAs, depender packaged transmissible RNAs, simple viruses and complicated viruses like the herpesviridae and poxviridae. Organelles and obligate cellular bacterial parasites like rickettsiae would seem to represent a different lineage completely (because of the independent capacity for protein synthesis).


 

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