Molecular and Fitness Barriers to Bunyavirus Reassortment

项目摘要/摘要 布尼亚病毒目(布尼亚病毒目)包括重要的人类、动物和植物病原体。和所有人一样 节段性病毒，重新排序是本亚病毒进化的主要驱动力。重新排序会产生病毒 带有不良表型，包括感染新宿主的能力。随着世界变得越来越 相互联系和被气候变化破坏，以前分离的布尼亚病毒有机会相遇 而且重新调整的情况正在增加。 两种病毒的蛋白质和RNA之间的相容性是它们能否产生 可存活的重组后代。而且，因为重新排序将不适应工作的蛋白质和RNA连接在一起 当与父母和其他人竞争时，新的重组人共同面临着一场艰难的进化之战 病毒在人群中传播。这一提议将改进对分子的机械理解。 阻碍重组，并研究允许重组出现的进化路径，尽管 最初的体能劣势。 我们已经设计了一种新的系统，它使用竞争微型基因组的文库来量化重新排序 同时大量病毒之间的潜力，并定义在案例中的分子崩溃 当他们做不到的时候。结合传统技术，我们建议使用这些“微型基因组混战”， 有针对性地回答有关布尼亚病毒之间的分子兼容性的问题。我们的团队结合了 在病毒基因组学、分子病毒学、蚊子感染和病毒进化方面的专业知识。 在目标1中，我们确定了当布尼亚病毒复制蛋白被破坏时病毒生命周期中的步骤 不匹配并执行定向进化实验，迫使不匹配的蛋白质适应工作 在一起。这将解释布尼亚病毒重组的一个关键限制因素，并详细说明 是布尼亚病毒生命周期的核心。在目标2中，我们使用我们的微型基因组混战系统来测试假设 正丁型病毒的重组相对不受包装的限制。在目标3中，我们检验了假设 与蚊子复制相关的瓶颈使不太适合的重新配型基因在 种群通过遗传漂移的随机效应。 在这个项目结束时，我们希望对分子有一个更好的理解。 确定两种布尼亚病毒是否可以重新组合的规则，重新组合的适合性后果，以及 重组病毒出现的进化途径。我们的结果可以用来参数化 预测布尼亚病毒出现风险的模型，并将阐明可能 是抗病毒药物的靶点。微型基因组杂交法原则上可以用来研究小鼠的生物学和进化。 各种RNA病毒，我们希望这项工作将建立一个广泛有用的平台。

Project Summary / Abstract The bunyavirus order (Bunyavirales) includes significant human, animal, and plant pathogens. As with all segmented viruses, reassortment is a major driver of bunyavirus evolution. Reassortment can produce viruses with undesirable phenotypes, including the ability to infect new hosts. As the world becomes increasingly interconnected and disrupted by climate change, the opportunity for previously isolated bunyaviruses to meet and reassort is increasing. Compatibility between the proteins and RNAs of two viruses is a key determinant of whether they can produce viable reassortant progeny. And, because reassortment joins proteins and RNAs that have not adapted to work together, new reassortants face an uphill evolutionary battle when competing with their parents and other viruses in the population. This proposal will generate an improved mechanistic understanding of molecular barriers to reassortment and investigate evolutionary pathways that permit reassortants to emerge despite initial fitness disadvantages. We have devised a new system that uses libraries of competing minigenomes to quantify reassortment potential between large numbers of viruses simultaneously, and to define the molecular breakdown in cases when they can’t. Using these “minigenome melees” in concert with traditional techniques, we propose to answer targeted questions about the molecular compatibility between bunyaviruses. Our team combines expertise in viral genomics, molecular virology, mosquito infection, and virus evolution. In aim 1, we identify steps in the viral lifecycle that break down when bunyavirus replication proteins are mismatched and perform directed evolution experiments that force mismatched proteins to adapt to work together. This will explain a key constraint on bunyavirus reassortment and detail molecular interactions at the heart of the bunyavirus life cycle. In aim 2, we use our minigenome melee system to test the hypothesis that orthobunyavirus reassortment is relatively unconstrained by packaging. In aim 3, we test the hypothesis that bottlenecks associated with replication in mosquitoes enable less fit reassortant genotypes to gain a foothold in populations via the stochastic effect of genetic drift. At the conclusion of this project, we expect to have a substantially improved understanding of the molecular rules that determine whether two bunyaviruses can reassort, the fitness consequences of reassortment, and the evolutionary pathways by which reassortant viruses emerge. Our results could be used to parameterize models that predict bunyavirus emergence risk and will shed light on conserved interactions that could be targeted by antiviral drugs. Minigenome melees could in principle be used to study the biology and evolution of all kinds of RNA viruses, and we expect this work to establish this as a broadly useful platform.