Functional and genetic constraints on influenza virus replication and fidelity

流感病毒的快速进化导致疫苗效力降低、间歇性耐药性以及新的季节性和大流行毒株的出现。流感病毒聚合酶复合体是甲型流感病毒(IAV)进化的核心，因为它是适应新宿主的主要因素，其复制保真度决定了病毒获得突变导致宿主范围扩大、耐药性或抗原漂移的速度。该项目的长期目标是阐明产生新病毒变异的机制，这对于了解病毒是如何出现和传播至关重要的。这个项目的目标是确定竞争的选择性力量如何驱动IAV聚合酶的进化。中心假设是复制速度和保真度之间存在内在冲突，IAV聚合酶的进化受到其RNA依赖的RNA聚合酶(RdRp)PB1高突变率的高度限制。该项目将应用系统发育分析、突变率分析、深度突变扫描(DMS)、分子动态模拟和体外聚合酶分析来确定IAV聚合酶的结构和功能约束。初步数据证实了这种方法的可行性，这些数据表明：(1)系统发育学方法可以定义IAV聚合酶在适应人类宿主时所采取的突变途径；(2)深度突变扫描(DMS)可以系统地定义氨基酸替换对聚合酶功能的影响；(3)一种新型的波动测试提供对每个核苷酸替换类的突变率的精确测量；(4)分子动力学建模和生物化学相结合，可以定义聚合酶异三聚体内部在功能上的重要相互作用。对IAV聚合酶的详细分析将通过三个目标完成。(目标1)定义在H3N2进化的50年中复制速度和保真度之间的权衡。竞争分析和波动测试将被用来确定在PB1的自然进化中出现的适应性突变的功能影响。(目的2)测量流感病毒RdRp中所有氨基酸突变对结构和功能的影响。PB1蛋白在没有和存在诱变核苷的情况下连续传代的DMS将用于评估每个突变对病毒复制和变异率的影响。(目标3)确定聚合酶复合体内的氨基酸相互作用如何影响复制和保真度。动态建模和体外分析将被用来机械地询问PB2、PB1和PA中共同选择的突变如何决定突变率。这项工作是创新的，因为它独特地结合了一系列互补的方法来测试关于病毒聚合酶进化的新假说。这项拟议的研究意义重大，因为它将定义对复制率和保真度的选择如何影响流感病毒的短期和长期进化。

The rapid evolution of influenza viruses has led to reduced vaccine efficacy, episodic drug resistance, and the emergence of novel seasonal and pandemic strains. The influenza virus polymerase complex is central to the evolution of influenza A viruses (IAV), as it is a major factor in adaptation to new hosts, and its replicative fidelity determines the rate at which the virus will acquire mutations that lead to host range expansion, drug resistance, or antigenic drift. The long-term goal of this project is to elucidate the mechanisms through which novel viral variants are generated, which is critical to understanding how viruses emerge and spread. The objective of this project is define how competing selective forces drive the evolution of the IAV polymerase. The central hypothesis is that there is an inherent conflict between replication speed and fidelity, and that the evolution of the IAV polymerase is highly constrained by the high mutation rate of PB1, its RNA-dependent RNA polymerase (RdRp). This project will apply phylogenetic analysis, mutation rate assays, deep mutational scanning (DMS), molecular dynamic modeling, and in vitro polymerase assays to define the structural and functional constrains on the IAV polymerase. The feasibility of this approach is supported by preliminary data, which show that: (i) phylogenetic approaches can define the mutational pathway taken by the IAV polymerase as it adapts to human hosts; (ii) deep mutational scanning (DMS) can systematically define the impact of amino acid substitutions on polymerase function; (iii) a novel fluctuation test provides precise measurements of mutation rates for each nucleotide substitution class; (iv) the combination of molecular dynamic modeling and biochemistry can define functionally important interactions within the polymerase heterotrimer. Detailed analyses of the IAV polymerase will be accomplished in three aims. (Aim 1) Define the trade-off between replicative speed and fidelity over 50 years of H3N2 evolution. Competition assays and fluctuation tests will be used to determine the functional impact of adaptive mutations that have arisen in the natural evolution of PB1. (Aim 2) Measure the structural and functional impacts of all amino acid mutations in the influenza virus RdRp. DMS of the PB1 protein with serial passage in the absence and presence of mutagenic nucleosides will be used to evaluate the impact of each mutation on viral replication and mutation rates. (Aim 3) Define how amino acid interactions within the polymerase complex affect replication and fidelity. Dynamic modeling and in vitro assays will be used to mechanistically interrogate how co-selected mutations in PB2, PB1 and PA determine mutation rate. This work is innovative, because it uniquely combines a range of complementary approaches to test a novel hypothesis regarding the evolution of viral polymerases. The proposed research is significant, because it will define how selection on replication rate and fidelity shape the short- and long-term evolution of influenza viruses.