PSTV, viroids



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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 g 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.