Abram Quinn H, Matthews Lindsay A, Guarné Alba, Sagan Selena M
Department of Biochemistry, McGill University, Montreal, Quebec, Canada, H3A 1A3.
Department of Microbiology and Immunology, University of British Columbia, British Columbia, Canada, V6T 1Z3.
RNA. 2025 Jul 16;31(8):1139-1153. doi: 10.1261/rna.080342.124.
Flavivirus infections, including those of Dengue virus (DENV) and Zika virus (ZIKV), result in a high disease burden globally, yet many aspects of their viral life cycle remain poorly understood. For example, while some features of the mechanism of negative-strand RNA synthesis are known, relatively little is known about the initiation of positive-strand RNA synthesis in the flavivirus life cycle. Viral RNA replication is initiated via the recruitment of the viral NS5 RNA-dependent RNA polymerase (RdRp) to stem-loop A (SLA) at the 5' terminus of positive-strand genomic RNA. Subsequent genome cyclization is thought to facilitate loading of NS5 onto the 3' terminus of the genomic RNA to initiate negative-strand RNA synthesis. Conversely, it is not clear whether RNA structures in the negative-strand replicative intermediate similarly recruit NS5 to promote positive-strand RNA synthesis, providing specificity to this process. Herein, we characterized the secondary structure of the 3' terminus of the negative-strand replicative intermediate in ZIKV and DENV1-4 in silico and in vitro. We observed that the 3' terminus of the negative strand is capable of forming a secondary structure which mirrors SLA, which we term SLA'. While we demonstrate that SLA' forms in vitro and is capable of interacting with NS5, introduction of G·U wobble base pairs that disrupt SLA', while keeping SLA largely intact, suggest that SLA' is not necessary for viral RNA replication. As such, this work suggests that in contrast to related viruses, the positive-strand promoter is unlikely to be provided by specific structure(s) at the 3' terminus of the negative-strand replicative intermediate.
黄病毒感染，包括登革热病毒（DENV）和寨卡病毒（ZIKV）感染，在全球造成了沉重的疾病负担，但其病毒生命周期的许多方面仍知之甚少。例如，虽然负链RNA合成机制的一些特征已为人所知，但关于黄病毒生命周期中正链RNA合成的起始却知之甚少。病毒RNA复制是通过将病毒NS5 RNA依赖性RNA聚合酶（RdRp）招募到正链基因组RNA 5'末端的茎环A（SLA）上启动的。随后的基因组环化被认为有助于将NS5加载到基因组RNA的3'末端以启动负链RNA合成。相反，尚不清楚负链复制中间体中的RNA结构是否同样招募NS5来促进正链RNA合成，从而为这一过程提供特异性。在此，我们在计算机模拟和体外实验中对寨卡病毒以及登革热病毒1 - 4型负链复制中间体3'末端的二级结构进行了表征。我们观察到负链的3'末端能够形成一种与SLA镜像的二级结构，我们将其称为SLA'。虽然我们证明SLA'在体外形成且能够与NS5相互作用，但引入破坏SLA'而使SLA基本保持完整的G·U摆动碱基对表明，SLA'对于病毒RNA复制并非必需。因此，这项研究表明，与相关病毒不同，正链启动子不太可能由负链复制中间体3'末端的特定结构提供。
