Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, College Park, Maryland, United States of America.
College of Plant Protection, Shandong Agricultural University, Taian, Shandong Province, P.R.China.
PLoS Pathog. 2018 Nov 19;14(11):e1007459. doi: 10.1371/journal.ppat.1007459. eCollection 2018 Nov.
Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3' untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3' UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3' UTR. Several additional carmovirus 3' UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5' end of their 3' UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3' UTRs.
无意义介导的衰变 (NMD) 是一种宿主 RNA 控制途径，可去除由于突变或剪接缺陷而产生的具有长 3'非翻译区 (UTR) 的异常转录本，这些转录本具有过早终止密码子 (PTC)。为了最大限度地发挥编码潜力，RNA 病毒通常含有内部终止密码子，这些密码子也应该是 NMD 的主要靶标。我们使用基于 agroinfiltration 的 NMD 测定法在 Nicotiana benthamiana 中鉴定了两个赋予 carmovirus Turnip crinkle virus (TCV) 基因组 NMD 抗性的片段。TCV p28 终止密码子下游的核糖体通读结构稳定了 NMD 敏感报告基因，umbravirus Pea enation mosaic virus 的移码元件也是如此。此外，TCV 3'UTR 起始处的 51-nt 无结构区 (USR) 插入到不相关的 NMD 敏感 3'UTR 中时，将 NMD 抗性提高了 3 倍。尽管类似区域没有序列相似性，但几个额外的 carmovirus 3'UTR 也根据构建体赋予了不同程度的 NMD 抗性。该 USR 下游的 2-nt 突变会显著增加 USR 中的二级结构，从而使 NMD 抗性略有降低，但当引入 USR 下游的 2-nt 突变时，抗性会被消除。该 2-nt 突变还增强了独立于基因组 RNA 表达的亚基因组 RNA 的 NMD 易感性。大多数 carmoviruses 在其 3'UTR 的 5'端缺乏 RNA 结构，这可能有助于增强亚基因组 RNA 的稳定性，从而增加编码衣壳蛋白的表达，衣壳蛋白也作为 RNA 沉默抑制剂发挥作用。这些结果表明，TCV 基因组具有固有的 NMD 抗性特征，这些策略可能在大多数 RNA 病毒和具有长 3'UTR 的 NMD 抗性宿主 mRNAs 中广泛存在。
