Viroids. NOMENCLATURE Replication Pathogenicity. Nabil Killiny

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1 Viroids NOMENCLATURE Replication Pathogenicity Nabil Killiny

2 A VIROID is A VIR(virus) OID(like) particle. Viroids are sub-viruses composed exclusively of a single circular strand of nucleic acid (RNA)

3 Very small, covalently closed, circular RNA molecules capable of autonomous replication and induction of disease. Range in size from approximately 20 nm No coding capacity - do not program their own polymerase. Use host-encoded polymerase for replication. Mechanically transmitted; often seed transmitted. More than 40 viroid species and many variants have been characterized. Classical viroids have been found only in plants. Viroids differ from viruses in that viruses, at their most basic level, consist of genetic material (DNA or RNA) contained within a protective protein shell. Viroids differ from prions, another type of subviral infectious agent, in that prions are made only of protein, lacking nucleic acid. HOW VIROID PROTECT ITSELF FROM nucleases? Host Specificity?

4 The smallest viroid identified so far is a 220 nucleobase scrna (small cytoplasmic RNA) associated with the rice yellow mottle sobemovirus(rymv) (Collins etal.1998). In comparison, the genome of the smallest known viruses capable of causing an infection by themselves are around two kilo bases in size. Many viroids consist of only 300 to 400 nucleotides. are Viroids a true pathogens?

5 DISCOVERY 1960s Raymer and O Brien develop a bioassay for the agent causing potato spindle tuber disease 1965 NOT Raymer teams up with Diener; they show that agent is not a typical virion 1968 Characterization of chrysanthemum stunt and citrus exocortis as nontypical viruses 1971 Diener demonstrates that the agent is a free noncoding RNA, coins the term viroid 1973 Electron micrograph shows viroid s hairpin structure Discovery 1974 Confirmation that viroids are non- coding 1976 EM shows that viroids form closed circular RNAs 1978 PSTVd is sequenced

6 NOMENCLATURE Viroid = virus- like

7 VIROID DISEASES Citrus exocortis viroid (CEVd) Causes stunting of plants, shelling of bark May result in little yield loss May be useful to promote dwarfing for agronomic advantage Transmitted through stock, graft Control by removal of infected plants, clean stock Citrus exocortis viroid

8 VIROID DISEASES CONTROL 1- Disinfection of cutting tools 2- Cold treatment e.g storage at 4 C for 6months or more, followed by apical shoot-tipculture, grafting, can be used to eliminate CSVd and HSVd. 3- Pre-inoculation with protective mild strains of viroid has proved effective to control PSTVd. HOW and HOW?

9 TRANSMISSION Most mechanically transmitted Some seed or pollen transmitted Tomato planta macho viroid-aphid transmitted (Ricardo Flores et al., 2005)

10 Viroid pathogenesis

11 Viroids and RNA silencing The evidence indicates that when viroids replicate via a double stranded intermediate RNA, they are targeted by a dicer enzyme and cleaved into sirnas that are then loaded onto the RNA-induced silencing complex. The viroid sirnas actually contain sequences capable of complementary base pairing with the plant's own messenger RNAs and induction of degradation or inhibition of translation is what causes the classic viroid symptoms.

12 Not all viroids are known to be pathogenic, but some are serious pathogens of plants. Viroids are usually transmitted by seed or pollen, but may be transported by farm implements as well. Infected plants can show distorted growth and sometimes are killed by the viroid.

13 VIROID STRUCTURES Two main groups of viroids: Self-cleaving Non-self- cleaving Non-self cleaving viroids replicate in nucleus and fold into dog bone or rod-like structure All are covalently closed circular RNAs fold to tightly basepaired structures Five domains identifiable in non-self-cleaving Terminal left (TL) Terminal right (TR) Pathogenicity (P) Central (C) Variable (V)

14 VIROID STRUCTURES The closed single-stranded RNA circle has extensive intrastrand base pairing and interspersed unpaired loops. Viroids have five domains. Most changes in viroid pathogenicity seem to arise from variations in the P and TL domains.

15 SCHEMATIC MODELS OF VIROID STRUCTURES (A) Rod-like secondary structure proposed for PSTVd, the type member of family Pospiviroidae. (B) Quasi rod-like secondary structure proposed for ASBVd, the type member of family Avsunviroidae. (C) Complex branched conformation proposed for PLMVd.

16 VIROID MOVEMENT Intra cellular movement cell-to-cell through plasmodesmata long distance through phloem

17 Host factors responsible for viroid movement Cellular proteins Phloem proteins- Phloem lectin PP2 Viroid binding protiens-virp1 Specific sequence or Structural motifs.

18 Viroid replication Viroid RNA does not code for any known protein; some even lack the AUG initiation codon. Nonetheless, they replicate autonomously in host cells. The replication mechanism involves interaction with RNA polymerase II, an enzyme normally associated with synthesis of messenger RNA, and "rolling circle" synthesis of new RNA.

19 Viroid replication Viroids replicate autonomously by using host-encoded RNA polymerase Concatemer: Multiple copies of a RNA sequence arranged end to end in tandem. Asymmetric rolling circle replication Member of Pospiviroidae replicate via an asymmetric Incoming (+)-circular RNA initially is transcribed into concatemeric linear (-)- strand RNA Which then serves as the replication intermediate for the synthesis of concatemeric, linear (+)- strand RNA This (+)- strand RNA subsequently is cleaved into unit length monomers that are ligated into circles

20 Enzymes involved in replication RNA polymerase RNA cleavage RNA ligase

21 Asymmetric Symmetric Asymmetric vs Symmetric

22 Symmetric rolling circle replication Member of Avsunviroidae replicate via an symmetric The circular (+)-RNA is transcribed into linear, concatemeric (-)- strand RNA Instead of serving as the direct template for the synthesis of linear concatemeric (+)- strand RNA The concatemeric (-)- strand RNA is cleaved into unit length molecules followed by circularization The circular (-)- RNA then serves as the template for the synthesis of linear, concatemeric (+)- strand RNA When subsequently is cleaved into unit-length monomers and circularized

23 Asymmetric Symmetric Asymmetric vs Symmetric