Dynamic regulation of RNA in the Zika virus life cycle

寨卡病毒生命周期中 RNA 的动态调控

• 批准号: RGPIN-2020-04713
• 负责人: Sagan, Selena
• 金额: $ 3.06万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740139
• 关键词: Dynamic; regulation; RNA; Zika; virus

RNA is arguably the most versatile biological macromolecule owing to its ability to both encode genetic information and to fold back on itself to form complex three-dimensional structures capable of highly specific ligand recognition and catalysis. RNA structure is also dynamic, meaning that a structure that plays an important role in one biological state may not persist under different biological conditions. Thus, studying RNA structure and organization is crucial to understanding its biological function. RNA secondary and higher ordered structures are also known to play a fundamental role in the life cycle of RNA viruses. Specific viral RNA structures have been demonstrated to act in a cell-type and species-specific manner to regulate the life cycle of mosquito-borne RNA viruses, such as Zika virus (ZIKV). We hypothesize that the ZIKV genomic RNA forms multiple dynamic structures that are critical for the viral life cycle. To test this hypothesis, we will perform Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) analysis on ZIKV RNAs in several biological contexts to identify RNA structures important for the viral life cycle. The following specific aims will be explored: 1. Characterization of the ZIKV genomic RNA in viral particles. To date, no viral packaging signals have been identified for ZIKV. In this aim, we will combine in vitro and in virio SHAPE analysis with photo-activatable cross-linking (PAR-CL). We anticipate that this will help define packaging signals and reveal the complex structure of the ZIKV genome inside viral particles. 2. Characterization of the dynamic structure of the ZIKV genomic RNA throughout the life cycle in human and mosquito cells. We will perform SHAPE analysis on viral RNAs stalled in one or more stages of the viral life cycle to generate a picture of the dynamics of RNA structure throughout the life cycle in human and mosquito cells. Identified regulatory motifs will be subject to mutagenesis and effects on viral translation, replication, and particle production will be assessed as well as the differential requirements between hosts. 3. Characterization of the negative-strand intermediate. To date, no studies have explored the structure of the negative-strand RNA intermediate. To identify the RNA structures important for positive-strand RNA synthesis, we will combine RNA structure prediction with SHAPE analysis of the negative-strand RNA to reveal insight into the requirements for positive-strand RNA synthesis. The proposed research is highly significant as it will improve our understanding of ZIKV biology. Characterization of novel RNA regulatory motifs will allow us to define new regulatory elements and refine models for viral translation, replication, and packaging. Moreover, identifying differential requirements in human vs. mosquito cells will provide insight into ZIKV evolution, adaptation, and epidemiological fitness.

RNA可以说是最通用的生物大分子，因为它能够编码遗传信息并折叠回自身以形成能够高度特异性配体识别和催化的复杂三维结构。RNA结构也是动态的，这意味着在一种生物状态中起重要作用的结构可能不会在不同的生物条件下持续存在。因此，研究RNA的结构和组织对于理解其生物学功能至关重要。 RNA的二级和高级结构也被认为在RNA病毒的生命周期中起着重要作用。已证明特定的病毒RNA结构以细胞类型和物种特异性方式起作用，以调节蚊媒RNA病毒（如寨卡病毒（ZIKV））的生命周期。我们假设ZIKV基因组RNA形成对病毒生命周期至关重要的多种动态结构。为了验证这一假设，我们将在几种生物学背景下对ZIKV RNA进行引物延伸（SHAPE）分析的选择性2'羟基酰化，以鉴定对病毒生命周期重要的RNA结构。将探讨以下具体目标：1.病毒颗粒中ZIKV基因组RNA的表征：迄今为止，尚未鉴定出ZIKV的病毒包装信号。在这个目标中，我们将结合联合收割机在体外和在virio形状分析与光活化交联（PAR-CL）。我们预计这将有助于定义包装信号并揭示病毒颗粒内ZIKV基因组的复杂结构。2. ZIKV基因组RNA在人类和蚊子细胞中整个生命周期的动态结构表征我们将对在病毒生命周期的一个或多个阶段停滞的病毒RNA进行SHAPE分析，以生成人类和蚊子细胞中整个生命周期的RNA结构动态图。将对已鉴定的调控基序进行诱变，并评估对病毒翻译、复制和颗粒产生的影响以及宿主之间的差异要求。3.负链中间体的表征。迄今为止，还没有研究探索负链RNA中间体的结构，为了确定对正链RNA合成重要的RNA结构，我们将联合收割机RNA结构预测与负链RNA的SHAPE分析相结合，以揭示正链RNA合成的要求。这项研究非常重要，因为它将提高我们对ZIKV生物学的理解。新的RNA调控基序的表征将使我们能够定义新的调控元件，并完善病毒翻译，复制和包装的模型。此外，识别人类与蚊子细胞中的差异需求将提供对ZIKV进化、适应和流行病学适应性的深入了解。