Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China.
J Virol. 2020 Feb 28;94(6). doi: 10.1128/JVI.01175-19.
Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation. Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.
蚊媒黄病毒由正链基因组 RNA 组成，两侧为非翻译区 (UTR)。UTR 中存在一系列具有关键功能的高度复杂的 RNA 结构。例如，Xrn1 抗性 RNA (xrRNA) 阻止 Xrn1 消化，导致亚基因组黄病毒 RNA (sfRNA) 的产生。保守的短直接重复序列 (DRs)，也称为保守序列 (CS) 和重复保守序列 (RCS)，已被鉴定为位于 xrRNA 下游的 RNA 元件之一，但它们的生物学功能仍然未知。在这项研究中，我们揭示了这些特定的 DRs 参与了哺乳动物和蚊子细胞中特定 sfRNA 的产生。生化分析和结构重塑表明，这些 DRs 茎部的碱基配对通过维持相应 xrRNA 与 Xrn1 的结合亲和力来控制 sfRNA 的形成。基于这些发现，我们提出 DRs 的功能类似于一个支架，保持 Xrn1-xrRNA 复合物的结合，从而促进 sfRNA 的形成。黄病毒包括许多重要的人类病原体。亚基因组黄病毒 RNA (sfRNA) 的产生是黄病毒的一个共同特征，对于病毒的致病性很重要。sfRNA 是通过细胞质 5'-3'外切核酸酶 Xrn1 在 3'-UTR 内的 Xrn1 抗性 RNA (xrRNA) 结构处不完全降解病毒基因组 RNA 形成的。黄病毒基因组的 3'-UTR 还包含独特的短直接重复序列 (DR)，如 RCS3、CS3、RCS2 和 CS2。然而，这些古老的初级 DR 序列的生物学功能在很大程度上仍然未知。在这里，我们发现 DR 序列参与 sfRNA 的形成和病毒的毒力，并为合理设计活减毒黄病毒疫苗提供了新的靶标。
