The 3'-terminal stem-loop (3'SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3'SL was proposed to act as a replication silencer. The lower part of the 3'SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3'SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3'SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3'SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3'SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3'SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

中文翻译：

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西尼罗河病毒基因组3'末端茎环元件的RNA温度计活性可调节宿主切换过程中的病毒复制效率。

黄病毒西尼罗河（WNV）的RNA基因组的3'-末端茎环（3'SL）在其茎中具有基因组环化所需的序列元件之一。由于环化是启动病毒复制的前提条件，因此有人提议将3'SL用作复制沉默子。3'SL的下部是亚稳的，具有结构灵活性，可调节病毒RNA从线性构象到环状构象的转换。在人类系统中，我们先前证明了细胞RNA结合蛋白AUF1 p45使3'SL不稳定，暴露了环化序列，从而促进了黄病毒基因组环化和RNA复制。通过研究3'SL稳定性增强的突变RNA，我们显示亚稳态元件的特定构象是AUF1 p45的螺旋破坏性RNA分子伴侣活性以及AUF1 p45支持的RNA复制起始的精度和效率的关键决定因素。在人和蚊子细胞中增加稳定性的突变WNV复制子的研究表明，培养温度大大影响了病毒RNA变异体的复制效率，并证明了3'SL的沉默效应是温度依赖性的。此外，我们鉴定并表征了与AUF1 p45具有相似活性的蚊子蛋白。但是，由于发现蚊子蛋白的RNA重塑活性远低于人蛋白，因此可能是细胞蛋白介导的3'不稳定化 提示SL在蚊子细胞中效率较低。总之，我们的数据支持一个模型，其中3'SL充当RNA温度计，可在宿主切换期间调节黄病毒复制。