Fig 1.
Rolling cycle replication of viroids.
(A) Asymmetric rolling circle replication in the Pospiviroidae occurs in the nucleus. Circular (+)-strand genomic RNA is transcribed by DNA-dependent RNA polymerase II (Pol II) to produce linear (-)-strand concatemers, which serve as template for Pol II-catalyzed synthesis of linear (+)-strand concatemers. These are cleaved to unit-length monomers by RNase III and circularized by DNA Ligase I. (B) Symmetric rolling circle replication in the Avsunviroidae occurs in the chloroplast. Circular (+)-strand genomic RNA is transcribed by nuclear-encoded chloroplast RNA polymerase (NEP) to generate linear (-)-strand concatemers, which are cleaved to monomers by an encoded hammerhead ribozyme and circularized by a tRNA ligase. Circular (-)-strand monomers serve as template for NEP-catalyzed synthesis of linear (+)-strand concatemers, followed by a second ribozyme cleavage to form monomers, which are circularized by tRNA ligase.
Fig 2.
PCR amplification of (-)-strand PSTVd concatemers.
(A) Schematic diagram shows transcription of (+)-strand PSTVd monomer by Pol II to synthesize linear (-)-strand concatemer, followed by cDNA synthesis from (-)-strand RNA using Superscript IV reverse transcriptase (SS-IV-RT). Taq DNA polymerase was employed to perform PCR amplification of duplex DNA copies from the cDNA using four separate primer pairs, as shown. The nucleotide coordinates of forward (F) and reverse (R) primers are indicated. Primers can bind to the same sites in all monomers, but for simplification only one binding site is indicated for each. PCR reactions (1) and (3) yield products with primers included in the concatemers, while reactions (2) and (4) yield products with concatemers flanked by primers (indicated by an asterisk). (B) With 5'→3' exonuclease activity, Taq DNA polymerase is able to synthesize through downstream products (nick translation). (C) PCR products amplified by Taq DNA Polymerase using primer sets 1 through 4 were resolved by 1% agarose gel electrophoresis. Ladders of discrete products correspond to genome concatemers. Monomer, hexamer, and heptamer products are indicated by asterisks (yellow, red, and blue, respectively).
Fig 3.
PCR amplification of (-)-strand PSTVd dimers and identification of de novo mutations.
(A) Diagram showing Pol II transcription of (+)-strand PSTVd monomer to (-)-strand linear concatemer, cDNA synthesis from (-)-strand RNA by Superscript IV reverse transcriptase (SS-IV-RT), and PCR amplification of dimeric duplex DNA from the cDNA by Phusion DNA polymerase. Shared and unshared mutations in individual monomers are represented by red and yellow dots, respectively. Primer pair 2 (see Fig 2) was used to amplify PCR products containing PSTVd monomers and dimers. (B) Monomer and dimer products were separated by 1% agarose gel electrophoresis. Dimer bands were excised from the gel, purified, and cloned.
Table 1.
Frequency and type of unshared de novo mutations observed in PSTVd (-)-strand dimer clones.
Table 2.
Unshared de novo mutations identified in PSTVd (-)-strand dimers.
Fig 4.
Base substitutions introduced by Pol II in PSTVd.
Mutations were mapped to genomic (+)-strand PSTVd. Number of transition and transversion events observed by dimer sequencing across all three biological replicates are shown. Asterisk indicates none observed.
Fig 5.
Comparison of mutations identified from PSTVd (-)-strand dimer sequencing and deep sequencing variant progeny.
(A) Secondary structure of the (+)-strand PSTVd genome. Nucleotide coordinates are indicated and loops/bulges are numbered 1 to 27 (red). (B) Mutations were mapped to genomic (+)-strand PSTVd. Mutations identified from dimer sequencing are indicated to the outside of the PSTVd secondary structure, and mutations identified by deep sequencing PSTVd progeny populations are indicated to the inside. Progeny mutations shown were shared by more than 10 unique sequences per million reads. Blue arrows indicate base substitutions, red arrows indicate base insertions, and brown triangles indicate base deletions. A deletion of seven consecutive bases is indicated by a brown box. Numbers of occurrences are indicated in parentheses. The terminal conserved region (TCR) and central conserved region (CCR) are outlined by black boxes.
Table 3.
Mutation hotspots identified by deep sequencing progeny populations.