Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB T1K 3M4, Canada.
Viruses. 2024 Apr 28;16(5):695. doi: 10.3390/v16050695.
The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.
双顺反子病毒间区（IGR）IRES 采用了最精简的翻译起始机制：IRES 无需使用蛋白因子即可直接招募核糖体，并从非 AUG 密码子起始翻译。已鉴定出几种双顺反子病毒 IRES 亚型；通常，IRES 采用两个至三个重叠的假结，具有关键的茎环和未配对区域，与核糖体 40S 和 60S 亚基的特定结构域相互作用，以指导翻译。我们之前预测了粉虱相关双顺反子病毒 2（BaDV-2）基因组中 IGR 的一种非典型结构和潜在的-1 程序性移码（-1 FS）信号。在这里，我们使用双顺反子报告基因，证明预测的 BaDV-2 -1 FS 信号可通过滑动序列和下游茎环结构在体外驱动-1 移码，从而指导病毒 RNA 依赖性 RNA 聚合酶的翻译。此外，预测的 BaDV-2 IGR 可在体外支持 IRES 翻译，但采用的机制与已知无因子双顺反子病毒 IGR IRES 机制不同。通过缺失和突变分析，BaDV-2 IGR IRES 定位于 140 个核苷酸元件内，并从 AUG 密码子起始翻译。此外，IRES 不会直接与纯化的核糖体结合，并且分别对 eIF2 和 eIF4A 抑制剂 NSC1198983 和 hippuristanol 敏感，表明这是一种 IRES 介导的因子依赖性机制。生物物理特性表明，BaDV-2 IGR IRES 包含几个茎环；然而，突变分析表明了一种模型，其中 IRES 无结构或为起始翻译采用不同的构象。总之，我们提供了该双顺反子病毒家族中第一个-1 FS 移码信号和新型因子依赖性 IRES 机制的证据，从而突出了病毒 RNA 结构策略的多样性，以指导病毒蛋白合成。
