PSTV, viroids

VIROIDS

References

Diener T.O. The viroid: Biological oddity or evolutionary fossil? Adv. Virus Res. 57, 137-184 (2001)
Ding, B., Kwon, M-Y. Hammond, R., Owens, R. Cell-to-cell movement of potato spindle tuber viroid. Plant J. 12, 931-36 (1997).
Hampel. A. The hairpin ribozyme; discovery, two-dimensional model, and development for gene therapy. Prog Nucl Acid Res and Mol Biol 58, 1-38 (1998)
Itaya, A., Matsuda, Y., Gonzales, R.A., Nelson, R.S., Ding, B. Potato spindle tuber viroid Strains of Different Pathogenicity Induces and Suppresses Expression of Common and Unique Genes in Infected Tomato. Mol. Plant Microbe Inter. 15, 990-999 (2002)
Lafontaine, D.A., Deschenes, P., Bussiere, F. Poisson, V., and Perreault, J-P. The viroid and viroid-like RNA database. Nucl Acids Res 27, 186-7 (1999) available online
Maniataki, E., Tabler, M., and Tsagris, M. Viroid RNA systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein VirP1 RNA 9, 346-54 2003
Owens, R.A., Steger, G., Hu, Y., Fels, A., Hammond, R.W., Riesner, D. RNA structural features responsible for potato spindle tuber viroid pathogenicity. Virol. 222, 144-158 (1996)
Pelchat, M., Grenier, C., Perreault, J-P. Characterization of a Viroid-Derived RNA Promoter for the DNA-Dependent RNA Polymerase from Escherichia coli, Biochem 41, 6561-71 (2002)
Symons, R.H. Plant pathogenic RNAs and RNA catalysis. Nucl Acids Res 25, 2683-89 (1997) available online
Symons, R.H., and Randles, J.W. Encapsidated circular viroid-like satellite RNAs (virusoids) of plants. Curr Topics Microbiol and Immunol 239, 81-105 (1999)
Zhu, Y., Qi, Y., Xun, Y., Owens, R., Ding, B. Movement of Potato Spindle Tuber Viroid Reveals Regulatory Points of Phloem-Mediated RNA Traffic Plant Physiol. 130 , 138–146 (2002)

Outline
I- Nature of viroids
1- Small RNA, 240-500 nucleotides, circular, highly base paired, extended conformation
2- Infectious, code for no proteins, rely on host polymerase
3- Virusoids are similar to viroids, but require helper virus
II- Viroid diseases
1- Potato spindle tuber group - pospiviroid
a- Citrus exocortis, serious disease, historically important
b- Coconut Cadang-cadang, a serious disease
2- "Virusoids" structurally similar to ASBV, but rely on viral polymerases, encapsidated by helper coat protein
III- PSTV - the first viroid to be physically characterized
1- A serious problem in potato propagation, known since 1922
2- Biological assays are inconvenient and often unreliable
3- Knife slashing transmits more efficiently than abrasion
4- May have arisen from a symptomless viroid in a solanaceous weed
IV- When do you suspect a viroid disease?
1- Mechanical transmissibility, but no virus particles
2- Infectivity not pelleted in the ultracentrifuge
3- Infectivity RNase sensitive
4- Viroid-like RNA detected by gel electrophoresis
V- Structure
1- Highly base paired; tissue contains both linear and circular forms which separate only on denaturing gels
2- Domain structure - based on base pairing predictions, chemical sensitivity and point mutations
a- Left terminal (T1)
b- Pathogenic (contains premelting region, hairpin II)
c- Central conserved (has intron boundary-like sequence)
d- Variable
e- Right terminal (T2), appears to mediate transport
3- Viroid melting is highly cooperative
VI- Isolation and assay of viroids
1- Diagnosis important in maintaining viroid free stock
2- Phenol extraction (normal RNA isolation procedure)
3- 2D gel electrophoresis, linear and circular forms comigrate under in native gels, circular forms retarded in second, denaturing dimension.
4- Return gel electrophoresis - native in forward direction until small RNAs leave gel, denatured (high temp) on return resolves viroids from host RNAs
5- Tissue usually contains about 1 µg viroid per gram of tissue
VII- Replication
1- Viroids depend on RNA polymerase II (nucleolus, α -amanitin sensitive)
2- Infection gives minus strand oligomers, suggests rolling circle replication
3- Oligomers cleave to give linear monomers
4- Ligases convert linear forms to circular. Linear forms generally contain terminal cyclic 2',3' phosphate, ligation converts to 2' phosphate
5- Virusoids cleave through "hairpin ribozyme"
VIII- How do viroids produce symptoms
1- PSTV variants breed true demonstrating self-coding
2- Symptom variants replicate normally showing that replication per se does not produce symptoms
3- Viroids which give different symptoms affect some genes in common and others uniquely (gene arrays)
IX- Viroid transport - probably requires binding to host protein(s)
1- Microinject viroid complexed to fluorescent dye
2- Viroid goes to nucleolus
3- Viroids move from cell to cell
4- Other sequences can "piggyback" on viroid
5- Since NAs by themselves don't enter nucleus, proteins are probably important.