Molecular and Fitness Barriers to Bunyavirus Reassortment

项目总结/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。如同所有 在节段病毒中，重组是布尼亚病毒进化的主要驱动力。基因重组可以产生病毒 具有不良表型，包括感染新宿主的能力。由于世界变得日益 相互联系并受到气候变化的破坏，以前孤立的布尼亚病毒有机会相遇， 并且重新排序正在增加。 两种病毒的蛋白质和RNA之间的相容性是决定它们能否产生 能存活的抗性后代。而且，由于重组连接了蛋白质和RNA， 新的繁殖体在与它们的父母和其他物种竞争时， 人群中的病毒。这一建议将产生一个改进的机制理解的分子 重组的障碍，并研究允许重组子出现的进化途径， 最初的健身劣势。 我们设计了一个新的系统，使用竞争的微型基因组库来量化重配 潜在的大量病毒之间的同时，并确定分子分解的情况下， 当他们不能。使用这些“微型基因组测序”与传统技术相结合，我们建议 回答有关布尼亚病毒之间分子相容性的有针对性的问题。我们的团队结合了 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aim 1中，我们确定了病毒生命周期中的步骤，当布尼亚病毒复制蛋白被破坏时， 并进行定向进化实验，迫使错配的蛋白质适应工作 一起这将解释布尼亚病毒重配的一个关键限制，并详细说明在病毒发生过程中的分子相互作用。 布尼亚病毒生命周期的核心在aim 2中，我们使用我们的迷你基因组混战系统来测试假设， 正布尼亚病毒重配相对不受包装的限制。在目标3中，我们检验假设， 与蚊子中的复制相关的瓶颈使得不太适合的抗性基因型在蚊子中获得立足点， 种群通过遗传漂变的随机效应。 在这个项目的结论，我们希望有一个实质性的改善了解的分子 确定两种布尼亚病毒是否可以重组的规则，重组的适应性后果，以及 抵抗病毒出现的进化途径。我们的结果可以用于参数化 预测布尼亚病毒出现风险的模型，并将揭示保守的相互作用， 被抗病毒药物靶向。原则上，微型基因组测序可以用于研究生物学和进化， 所有类型的RNA病毒，我们希望这项工作能够将其建立为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.