Origin and Evolution of Defective Interfering RNAs of Tomato Bushy Stunt Virus

Knorr, David A.; Morris, T. Jack

doi:10.1007/978-1-4615-3304-7_6

Cited by 2 publications

(4 citation statements)

References 23 publications

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“…Accumulation and evolution of putative DI RNA precursors. Previous studies on tombusvirus DI RNA formation and evolution were unable to detect any molecules larger than 16,17,29). Presumably, larger precursors do exist but, for unknown reasons, cannot accumulate to detectable levels or are transient.…”

Section: Resultsmentioning

confidence: 87%

“…Factors influencing competitiveness of DI RNAs. We have shown previously that during high-multiplicity serial passage of TBSV in plants, a DI RNA species approximately 800 nucleotides (nt) long was the first dominant species to accumulate but, with continued passage, was replaced by a smaller, -600-nt DI RNA species (16). The fact that the smaller species was the dominant DI RNA later in the passage series suggested that it was more competitive (i.e., more readily able to accumulate) than its larger counterpart.…”

Section: Resultsmentioning

confidence: 99%

“…In the present study, we were interested in determining which of these DI RNA species is the more competitive and what factors influence the observed levels of competitiveness. CDNA copies corresponding to the larger (DI-73) and smaller (DI-72) DI RNA species had previously been cloned downstream of a T7 RNA polymerase promoter (16) and are shown schematically in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

“…HS-7 is an infectious clone of TBSV genomic RNA (gRNA) which does not express a functional viral coat protein (35) and was generously supplied by Herman Scholthof. The construction of plasmids DI-72 and DI-73, containing cloned cDNAs of naturally occurring DI RNAs, has already been described (16,17). DI-82 and DI-83 were constructed by digestion of TBSV-100 with BstXI (coordinate 1391) and SphI (in vector, 3' to the viral sequence) and replacement of the excised viral fragment with the corresponding BstXI and SphI fragments from DI-72 and DI-73, respectively.…”

Section: Materuils and Methodsmentioning

confidence: 99%

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

White

Morris

1994

J Virol

129

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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.

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Materuils and Methodsmentioning

confidence: 99%

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