Qiu W, Park J W, Jackson A O, Scholthof H B
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843, USA.
Virology. 2001 Mar 1;281(1):51-60. doi: 10.1006/viro.2001.0763.
Tomato bushy stunt virus (TBSV) and other tombusviruses are notorious for their propensity to accumulate defective interfering RNAs (DIs) upon serial passage through experimental Nicotiana species. Hallmarks of this occurrence include reduced levels of helper RNA and protein accumulation and amelioration of the lethal necrosis induced upon infection of the host with the helper viruses alone. The objective of this study was to determine whether the prolific trans-accumulation of defective RNAs typically occurs for all replicase-deficient TBSV mutants, or if this process is influenced by internal cis-acting elements that have been excised from DIs. For this purpose, various replicase-deficient TBSV cDNA constructs were generated and their transcripts were tested for trans-accumulation competence in the presence of helper virus. The results revealed that a region of ca. 150 nucleotides near the center of the replicase gene, with a predicted high degree of secondary structure, was a potent inhibitor of trans-rescue (ITR) by TBSV. Relocation of the ITR into efficiently trans-replicating DIs inhibited their accumulation drastically, but only when inserted in the reverse orientation and with an intact 5' ITR-specific predicted hairpin structure. Insertion of the ITR element in the positive orientation yielded DI transcripts that were able to replicate, but failed to interfere noticeably with either accumulation of the helper RNA or the onset of the lethal necrosis phenotype in N. benthamiana. In conclusion, the ITR has an intrinsic capacity to inhibit trans-accumulation of defective RNAs, but its stringency and biological effects are strongly influenced by the overall sequence context.
番茄丛矮病毒（TBSV）和其他番茄病毒因在通过实验烟草属植物连续传代时易于积累缺陷干扰RNA（DIs）而声名狼藉。这种现象的特征包括辅助RNA和蛋白质积累水平降低，以及单独用辅助病毒感染宿主时诱导的致死性坏死得到缓解。本研究的目的是确定缺陷RNA的大量反式积累是否通常发生在所有复制酶缺陷的TBSV突变体中，或者这个过程是否受已从DIs中切除的内部顺式作用元件的影响。为此，构建了各种复制酶缺陷的TBSV cDNA构建体，并在辅助病毒存在的情况下测试了它们的转录本的反式积累能力。结果表明，复制酶基因中心附近约150个核苷酸的区域，具有预测的高度二级结构，是TBSV反式拯救（ITR）的有效抑制剂。将ITR重新定位到高效反式复制的DIs中会显著抑制它们的积累，但只有当以反向插入且具有完整的5' ITR特异性预测发夹结构时才会如此。以正向插入ITR元件产生的DI转录本能够复制，但未能明显干扰辅助RNA的积累或本氏烟草中致死坏死表型的出现。总之，ITR具有抑制缺陷RNA反式积累的内在能力，但其严格性和生物学效应受到整体序列背景的强烈影响。
