Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20772, USA.
Viruses. 2023 Feb 27;15(3):638. doi: 10.3390/v15030638.
The cap-independent translation of plus-strand RNA plant viruses frequently depends on 3' structures to attract translation initiation factors that bind ribosomal subunits or bind directly to ribosomes. Umbraviruses are excellent models for studying 3' cap-independent translation enhancers (3'CITEs), as umbraviruses can have different 3'CITEs in the central region of their lengthy 3'UTRs, and most also have a particular 3'CITE (the T-shaped structure or 3'TSS) near their 3' ends. We discovered a novel hairpin just upstream of the centrally located (known or putative) 3'CITEs in all 14 umbraviruses. These CITE-associated structures (CASs) have conserved sequences in their apical loops and at the stem base and adjacent positions. In 11 umbraviruses, CASs are preceded by two small hairpins joined by a putative kissing loop interaction (KL). Converting the conserved 6-nt apical loop to a GNRA tetraloop in opium poppy mosaic virus (OPMV) and pea enation mosaic virus 2 (PEMV2) enhanced translation of genomic (g)RNA, but not subgenomic (sg)RNA reporter constructs, and significantly repressed virus accumulation in . Other alterations throughout OPMV CAS also repressed virus accumulation and only enhanced sgRNA reporter translation, while mutations in the lower stem repressed gRNA reporter translation. Similar mutations in the PEMV2 CAS also repressed accumulation but did not significantly affect gRNA or sgRNA reporter translation, with the exception of deletion of the entire hairpin, which only reduced translation of the gRNA reporter. OPMV CAS mutations had little effect on the downstream BTE 3'CITE or upstream KL element, while PEMV2 CAS mutations significantly altered KL structures. These results introduce an additional element associated with different 3'CITEs that differentially affect the structure and translation of different umbraviruses.
植物正链 RNA 病毒的帽非依赖性翻译通常依赖于 3' 结构来吸引结合核糖体亚基或直接结合核糖体的翻译起始因子。弹状病毒是研究 3' 帽非依赖性翻译增强子（3'CITE）的极好模型，因为弹状病毒在其长 3'UTR 的中心区域可以具有不同的 3'CITE，并且大多数还在其 3' 末端附近具有特定的 3'CITE（T 形结构或 3'TSS）。我们在所有 14 种弹状病毒中发现了一种新的发夹结构，它位于中央（已知或推测）3'CITE 上游。这些与 CITE 相关的结构（CAS）在其顶端环和茎基部以及相邻位置具有保守序列。在 11 种弹状病毒中，CAS 之前有两个由假定的亲吻环相互作用（KL）连接的小发夹。将鸦片烟花叶病毒（OPMV）和豌豆花叶卷曲病毒 2（PEMV2）中保守的 6 个核苷酸顶端环转换为 GNRA 四链体可增强基因组（g）RNA 的翻译，但不增强亚基因组（sg）RNA 报告基因构建体的翻译，并显著抑制病毒积累。OPMV CAS 中的其他改变也抑制了病毒积累，仅增强了 sgRNA 报告基因的翻译，而下茎中的突变则抑制了 gRNA 报告基因的翻译。PEMV2 CAS 中的类似突变也抑制了积累，但对 gRNA 或 sgRNA 报告基因的翻译没有显著影响，除了整个发夹缺失，这仅降低了 gRNA 报告基因的翻译。OPMV CAS 突变对下游 BTE 3'CITE 或上游 KL 元件几乎没有影响，而 PEMV2 CAS 突变则显著改变了 KL 结构。这些结果引入了与不同 3'CITE 相关的另一个元素，该元素可不同程度地影响不同弹状病毒的结构和翻译。
