Mapping epistasis in molecular evolution: the influenza nucleoprotein

描述(由申请人提供)： 当一个等位基因的效果受到另一个等位基因的影响时，上位性就会发生。上位性相互作用在进化中发挥着深远的作用。对上位性的理解在各种生物医学努力中也是至关重要的，例如从遗传关联研究中识别疾病变异和预测病原体的进化。但关于上位性的许多最基本的问题仍然没有得到回答，包括：上位性相互作用有多常见？它们是如何产生的？潜在的分子机制是什么？我们将通过绘制沿着真实进化轨迹的上位性相互作用来解决这些问题。为了做到这一点，我们将使用计算和实验工具的组合来研究流感核蛋白。由于流感进化的独特性质，我们能够一步一步地详细推断自1968年以来人类H3N2核蛋白发生的39个突变。我们将沿着这一进化轨迹构建所有39个中间蛋白质。我们还将把每个突变分别引入1968年的父母。所有这些变种都将接受生化功能和对病毒适应性的影响测试。这将为上位性提供一个明确的实验测试：如果一个突变在1968年的亲本中具有不同于它实际发生的进化中间阶段的影响，那么它就参与了上位性相互作用。至关重要的是，我们的初步工作已经确定了上位性相互作用中涉及的几个突变。在目标1中，我们将在这项工作的基础上绘制自1968年以来的所有上位性相互作用图。在目标2中，我们将解决上位性相互作用如何产生的谜团--是否同时发生多个突变，是否存在补偿性或允许性突变，或者上位性是由于逐渐变化的遗传背景而缓慢出现的？最后，在目标3中，我们将使用生物物理和生化技术来确定每个上位性相互作用的分子机制。在这项研究的结论中，我们将沿着真实的进化轨迹绘制出上位性相互作用的流行、起源和机制。我们的发现将为了解塑造蛋白质和病毒进化的最重要因素之一提供一个新的窗口，并将有助于尝试解释序列数据和理解等位基因之间的相互作用。

DESCRIPTION (provided by applicant): Epistasis occurs when the effect of one allele is influenced by another allele. Epistatic interactions play a profound role in evolution. An understanding of epistasis is also crucial in a variety of biomedical endeavors, such as identifying disease variants from genetic association studies and predicting the evolution of pathogens. But many of the most basic questions about epistasis remain unanswered, including: How common are epistatic interactions? How do they arise? And what are the underlying molecular mechanisms? We will address these questions by mapping epistatic interactions along a real evolutionary trajectory. To do this, we will employ a combination of computational and experimental tools to study the influenza nucleoprotein. Because of the unique nature of influenza evolution, we are able to infer in step-by-step detail the 39 mutations that have occurred in the nucleoprotein from human H3N2 since the year 1968. We will construct all 39 intermediate proteins along this evolutionary trajectory. We will also introduce each of the mutations individually into the 1968 parent. All of these variants will be tested for biochemical function and effect on viral fitness. This will provide a clear experimental test for epistasis: a mutation is involved in an epistatic interaction if it has a different effect in the 1968 parent than in the evolutionary intermediate in which it actually occurred. Crucially, our preliminary work has already identified several mutations involved in epistatic interactions. In Aim 1, we will build on this work by mapping all epistatic interactions since 1968. In Aim 2, we will address the mystery of how the epistatic interactions arose - did multiple mutations occur simultaneously, were there compensatory or permissive mutations, or did epistasis arise slowly due to a gradually shifting genetic background? Finally, in Aim 3, we will use biophysical and biochemical techniques to identify the molecular mechanisms of each epistatic interaction. At the conclusion of this study, we will have mapped the prevalence, origins, and mechanisms of epistatic interactions along a real evolutionary trajectory. Our findings will provide a new window into one of the most important factors shaping the evolution of proteins and viruses, and will aid in attempts to interpret sequence data and understand interactions between alleles.