Agharkar Research Institute, Nanobioscience Group, G. G. Agarkar Road, Pune 411004, India; Savitribai Phule Pune University, Ganeshkhind, Pune 411 007, India.
Agharkar Research Institute, Nanobioscience Group, G. G. Agarkar Road, Pune 411004, India; Savitribai Phule Pune University, Ganeshkhind, Pune 411 007, India.
J Mol Biol. 2023 May 15;435(10):168050. doi: 10.1016/j.jmb.2023.168050. Epub 2023 Mar 17.
In the present investigation, we have identified the functional significance of the highly conserved miR-140 binding site on the Hepatitis E Virus (HEV) genome. Multiple sequence alignment of the viral genome sequences along with RNA folding prediction indicated that the putative miR-140 binding site has significant conservation for sequence and secondary RNA structure among HEV genotypes. Site-directed mutagenesis and reporter assays indicated that an intact sequence of the miR-140 binding site is essential for HEV translation. Provision of mutant miR-140 oligos carrying same mutation as on mutant HEV successfully rescued mutant HEV replication. In vitro cell-based assays with modified oligos proved that host factor-miR-140 is a critical requirement for HEV replication. Biotinylated RNA pulldown and RNA immunoprecipitation assays proved that the predicted secondary RNA structure of the miR-140 binding site allows the recruitment of hnRNP K, which is a key protein of the HEV replication complex. We predicted the model from the obtained results that the miR-140 binding site can serve as a platform for recruitment of hnRNP K and other proteins of HEV replication complex only in the presence of miR-140.
在本研究中，我们确定了高度保守的 miR-140 结合位点在戊型肝炎病毒（HEV）基因组中的功能意义。病毒基因组序列的多序列比对和 RNA 折叠预测表明，假定的 miR-140 结合位点在 HEV 基因型之间具有序列和二级 RNA 结构的显著保守性。定点突变和报告基因实验表明，miR-140 结合位点的完整序列对于 HEV 翻译是必不可少的。携带与突变 HEV 相同突变的突变 miR-140 寡核苷酸的提供成功挽救了突变 HEV 的复制。具有修饰的寡核苷酸的体外细胞测定证明宿主因子-miR-140 是 HEV 复制的关键要求。生物素化 RNA 下拉和 RNA 免疫沉淀测定证明了 miR-140 结合位点的预测二级 RNA 结构允许 hnRNP K 的募集，hnRNP K 是 HEV 复制复合物的关键蛋白。我们根据获得的结果预测了模型，即只有在存在 miR-140 的情况下，miR-140 结合位点才能作为 hnRNP K 和 HEV 复制复合物其他蛋白募集的平台。
