Epistasis Within and Between Genomic Segments in Influenza Virus

项目总结/摘要 甲型流感病毒（IAV）在疫苗和药物的作用下迅速进化，造成重大的公共卫生和 经济负担。突变之间的相互作用，或上位性，决定了IAV将如何进化。不同类型 包括积极的和消极的上位性，有不同的进化后果。然而， 关于IAV的上位性类型，限制了我们预测和控制病毒的能力。长期目标是 阐明基本的进化过程如何影响公共卫生。该项目的目标是描述 IAV的所有8个基因组区段之间和编码抗原性的HA基因内的成对上位性 血凝素蛋白一个易于处理的遗传系统将允许创造成千上万的双突变体， 一个现有的具有确定点突变的流感病毒全基因组文库。用一种新颖、敏感、 在用于病毒生长的高通量适合性测定中，将确定每个单突变体和双突变体的适合性， 并且上位性的大小和符号将被量化。初步数据表明，这种适应性测定具有 非常低的测量误差，能够精确测量数千个突变体中的上位性。第一个目标 是表征所有IAV片段对之间的成对上位性。根据相关理论预测， 对于病毒进化的限制，假设IAV中的上位性平均为负。明显上位性 片段之间的模式可以揭示先前未知的IAV片段之间的功能相互作用。到 完成这一目标，数以千计的双和单突变体在所有成对的片段中的适应性将是 比较了第二个目的是表征HA内的成对上位性。基于以前对蛋白质的研究- 折叠限制，假设HA内的上位性平均为负。为了验证这一假设， 将比较HA内数百个双突变体和单突变体的适合度。这个项目是创新的，因为， 据我们所知，这将是唯一的研究之间的上位性随机点突变在不同基因的IAV 几乎是病毒研究的100倍。这种创新是由创造性的 使用IAV基因工程系统和一种新的适合度测定。这个项目意义重大，因为 描述IAV的上位性类型将改善对季节性和大流行性疾病演变的预测。 并帮助设计有效的疫苗。通过完成拟议的研究项目， 指导和其他活动，申请人将实现他在分子进化领域的培训目标 和群体遗传学，高通量方法和数据分析，有效的科学交流， 临床医学这将使申请人能够实现成为一名物理学家-科学家的最终目标 他回答了进化生物学中的基本问题，并对人类健康产生了实际影响。

Project Summary/Abstract Influenza A virus (IAV) evolves rapidly in response to vaccines and drugs, causing significant public health and economic burdens. Interactions between mutations, or epistasis, determine how IAV will evolve. Different types of epistasis, including positive and negative, have different evolutionary consequences. However, little is known about the types of epistasis in IAV, limiting our ability to predict and control the virus. The long-term goal is to elucidate how basic evolutionary processes affect public health. The objectives of this project are to characterize pairwise epistasis between all 8 genomic segments of IAV and within the HA gene encoding the antigenic hemagglutinin protein. A tractable genetic system will allow the creation of thousands of double mutants using an existing genome-wide library of influenza viruses with defined point mutations. Using a novel, sensitive, and high-throughput fitness assay for viral growth, the fitness of each single and double mutant will be determined, and the magnitude and sign of epistasis will be quantified. Preliminary data indicate that this fitness assay has very low measurement error, enabling precise measurement of epistasis in thousands of mutants. The first aim is to characterize pairwise epistasis between all pairs of IAV segments. Based on theoretical predictions related to viral evolutionary constraints, the hypothesis is that epistasis in IAV is negative on average. Distinct epistatic patterns between segments may reveal previously unknown functional interactions between IAV segments. To complete this aim, the fitness of thousands of double and single mutants in all pairs of segments will be compared. The second aim is to characterize pairwise epistasis within HA. Based on previous work on protein- folding constraints, the hypothesis is that epistasis within HA is negative on average. To test this hypothesis, the fitness of hundreds of double and single mutants within HA will be compared. This project is innovative because, to our knowledge, it will be the only study of epistasis between random point mutations in different genes of IAV and almost a hundred times larger than the largest such study in a virus. This innovation is enabled by creative use of the IAV genetic engineering system and a novel fitness assay. This project is significant because characterizing the types of epistasis in IAV will improve predictions of the evolution of seasonal and pandemic strains and aid in the design of effective vaccines. Through completion of the proposed research project, mentorship, and other activities, the applicant will achieve his training goals in the fields of molecular evolution and population genetics, high-throughput methods and data analysis, effective scientific communication, and clinical medicine. This will enable the applicant to achieve his ultimate goal of becoming a physician-scientist who answers fundamental questions in evolutionary biology with practical implications for human health.