Nonhomologous RNA recombination in tombusviruses: generation and evolution of defective interfering RNAs by stepwise deletions

Abstract

We used a protoplast system to study the mechanisms involved in the generation and evolution of defective interfering (DI) RNAs of tomato bushy stunt tombusvirus (TBSV). Synthetic transcripts corresponding to different naturally occurring TBSV DI RNAs, or to various artificially constructed TBSV defective RNAs, were analyzed. The relative levels of competitiveness of different DI RNAs were determined by coinoculating their corresponding transcripts into protoplasts along with helper genomic RNA transcripts and monitoring the level of DI RNA accumulation. Further studies were performed to assess the contribution of naked DI RNA stability and DI RNA encapsidation efficiency to the observed levels of competitiveness. In addition, the ability of various defective RNAs to evolve to alternative forms was tested by serially passaging protoplast infections initiated with transcripts corresponding to helper genomic RNA and a single type of defective RNA. These studies, and the analysis of the sequences of observed recombinants, indicate that (i) replication competence is a major factor dictating DI RNA competitiveness and is likely a primary determinant in DI RNA evolution, (ii) DI RNAs are capable of evolving to both smaller and larger forms, and the rates at which various transitions occur differ, (iii) DI RNA-DI RNA recombination and/or rearrangement is responsible for the formation of the evolved RNA molecules which were examined, and (iv) sequence complementarities between positiveand negative-sense strands in the regions of the junctions suggest that, in some cases, base pairing between an incomplete replicase-associated nascent strand and acceptor template may mediate selection of recombination sites. On the basis of our data, we propose a stepwise deletion model to describe the temporal order of events leading to the formation of tombusvirus DI RNAs.

Nucleotide-Resolution Profiling of RNA Recombination in the Encapsidated Genome of a Eukaryotic RNA Virus by Next-Generation Sequencing

“…37 Such a scenario is what we would expect during the generation of defective RNAs, which are initially generated by stochastic RNA recombination but are subsequently highly (sometimes competitively) replicated by viral RdRps. 12…”

“…11 Conversely, viral RNA recombination may also facilitate the stepwise generation of defective RNAs. 12 Defective RNAs have lost their ability to independently encode functional viral proteins but maintain the sequence motifs required for RNA replication by the viral RNA-dependent RNA polymerase (RdRp) and so are able to proliferate parasitically. Additionally, defective RNAs are often packaged into viral particles and so may contain the necessary motifs required for encapsidation.…”

Atypical RNA Elements Modulate Translational Readthrough in Tobacco Necrosis Virus D

“…Preparation of viral RNAs. Uncapped in vitro transcripts of SmaI-linearized TNV-D constructs were produced using the AmpliScribe T7-Flash transcription kit (Epicentre Technologies) as described previously (45). RNA transcript concentrations were quantified by spectrophotometry, and the quality of transcripts was verified by agarose gel electrophoresis.…”

“…Protoplast infection. Cucumber cotyledon protoplasts were prepared and transfected with uncapped in vitro transcribed viral genomic RNA (45). Specifically, 3 ϫ 10 5 protoplasts were transfected with 3 g of viral genomic RNA and incubated under constant light for 22 h at either 22 or 29°C.…”

“…Specifically, 3 ϫ 10 5 protoplasts were transfected with 3 g of viral genomic RNA and incubated under constant light for 22 h at either 22 or 29°C. The total nucleic acids were extracted as described previously (45) and separated in nondenaturing 2% agarose gels. Viral RNAs were detected via Northern blotting using three 32 P-radiolabeled probes complementary to the 3= end of TNV-D: nt 2821 to 2840, 3520 to 3532, and 3643 to 3663 (31).…”