White K A, Morris T J
School of Biological Sciences, University of Nebraska-Lincoln 68588-0118.
J Virol. 1994 Jan;68(1):14-24. doi: 10.1128/JVI.68.1.14-24.1994.
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 positive- and 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.
我们使用原生质体系统来研究番茄丛矮番茄病毒（TBSV）缺陷干扰（DI）RNA的产生和进化机制。分析了与不同天然存在的TBSV DI RNA相对应的合成转录本，或各种人工构建的TBSV缺陷RNA。通过将不同DI RNA的相应转录本与辅助基因组RNA转录本一起共接种到原生质体中，并监测DI RNA积累水平，来确定不同DI RNA的相对竞争水平。进一步的研究旨在评估裸露DI RNA稳定性和DI RNA衣壳化效率对观察到的竞争水平的贡献。此外，通过对由辅助基因组RNA和单一类型缺陷RNA的转录本引发的原生质体感染进行连续传代，测试了各种缺陷RNA进化为替代形式的能力。这些研究以及对观察到的重组体序列的分析表明：（i）复制能力是决定DI RNA竞争力的主要因素，并且可能是DI RNA进化的主要决定因素；（ii）DI RNA能够进化为更小和更大的形式，并且各种转变发生的速率不同；（iii）DI RNA-DI RNA重组和/或重排负责所检测到的进化RNA分子的形成；（iv）在连接区域正链和负链之间的序列互补性表明，在某些情况下，不完全复制酶相关新生链与受体模板之间的碱基配对可能介导重组位点的选择。基于我们的数据，我们提出了一个逐步缺失模型来描述导致番茄病毒DI RNA形成的事件的时间顺序。
