Palo Michael Z, Ha Betty, Lapointe Christopher P, Alvarado Carlos, Janetzko John, Carette Jan E, Puglisi Joseph D, Puglisi Elisabetta Viani
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, United States.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, United States.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf514.
Long-range tertiary interactions are a widespread structural feature in viral RNAs (vRNAs) and mRNAs. In the orthoflaviviruses, conserved complementary sequences in the 5' and 3' terminal regions have an essential role in viral replication. Long-range pairing of these conserved sequences is proposed to facilitate a switch between two alternative vRNA conformations. Yet the detailed nature of these interactions, their relative populations and their exchange are required to formulate a mechanistic model of their role in regulation of the viral life cycle. Here, we used single-molecule Förster resonance energy transfer to study the global conformation of vRNAs by measuring their end-to-end distances. We observed that vRNA conformation is heterogeneous, and that conformers with close end-to-end distances have unusual kinetic stability when compared with mRNA lacking these specific long-range interactions. vRNAs also partition between at least two stable states with a large rearrangement of the terminal regions (>50 Å change in end-to-end distance). We demonstrate that this bistability depends on long-range interactions and is modulated by host factors such as the initiation factor complex eIF4F. Understanding how vRNA and its stability is influenced by interactions with other host and viral factors will help to elucidate a mechanistic role for these highly conserved orthoflaviviral sequences.
远距离三级相互作用是病毒RNA（vRNA）和mRNA中广泛存在的结构特征。在正黄病毒中，5'和3'末端区域的保守互补序列在病毒复制中起着至关重要的作用。这些保守序列的远距离配对被认为有助于在两种替代vRNA构象之间进行转换。然而，需要这些相互作用的详细性质、它们的相对丰度及其交换情况，以构建一个关于它们在病毒生命周期调控中作用的机制模型。在这里，我们使用单分子Förster共振能量转移通过测量vRNA的端到端距离来研究其整体构象。我们观察到vRNA构象是异质的，并且与缺乏这些特定远距离相互作用的mRNA相比，端到端距离相近的构象异构体具有不同寻常的动力学稳定性。vRNA还在至少两种稳定状态之间分配，其末端区域有大量重排（端到端距离变化>50 Å）。我们证明这种双稳态取决于远距离相互作用，并受到宿主因子如起始因子复合物eIF4F的调节。了解vRNA及其稳定性如何受到与其他宿主和病毒因子相互作用的影响，将有助于阐明这些高度保守的正黄病毒序列的机制作用。
