Functional and genetic constraints on influenza virus replication and fidelity

流感病毒复制和保真度的功能和遗传限制

• 批准号: 10647866
• 负责人: Adam Lauring
• 金额: $ 41.04万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-17 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647866
• 关键词: Affect; Amino Acid Substitution; Amino Acids; Antiviral Agents; Biochemical; Biochemistry; Biological Assay; Birds; Codon Nucleotides; Complex; Conflict (Psychology); Data; Development; Drug resistance; Evolution; Genetic; Genome; Goals; Human; In Vitro; Infection prevention; Influenza; Influenza A Virus, H3N2 Subtype; Influenza A virus; Integration Host Factors; Kinetics; Libraries; Maps; Measurement; Measures; Modeling; Mutagens; Mutate; Mutation; Natural Selections; Nucleosides; Nucleotides; Pathway interactions; Pharmaceutical Preparations; Phylogenetic Analysis; Polymerase; Population; Property; Proteins; Public Health; RNA-Directed RNA Polymerase; Research; Resistance; Route; Seasons; Serial Passage; Shapes; Site; Speed; Testing; Variant; Viral; Virus; Virus Replication; Work; cost; fitness; in vitro Assay; influenzavirus; innovation; molecular dynamics; mutation screening; novel; outbreak control; pandemic disease; pressure; protein protein interaction; single-molecule FRET; transmission process; vaccine efficacy; viral RNA

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.

流感病毒的快速演变导致疫苗功效，情节性耐药性以及新型季节性和大流行菌株的出现。流感病毒聚合酶复合物对于流感病毒（IAV）的演变至关重要，因为它是适应新宿主的主要因素，其复制性保真度决定了该病毒将获取突变的速率，从而导致宿主范围扩张，耐药性或抗原漂移。该项目的长期目标是阐明产生新型病毒变异的机制，这对于了解病毒的出现和传播至关重要。该项目的目的是定义竞争性选择性如何驱动IAV聚合酶的演变。中心假设是复制速度和忠诚度之间存在固有的冲突，并且IAV聚合酶的演变受PB1高突变率（其RNA依赖性RNA聚合酶（RDRP））的高度限制。该项目将采用系统发育分析，突变率测定，深突变扫描（DMS），分子动力学建模和体外聚合酶测定，以定义对IAV聚合酶的结构和功能约束。初步数据支持这种方法的可行性，这些数据表明：（i）系统发育方法可以定义IAV聚合酶适应人类宿主的突变途径； （ii）深突变扫描（DMS）可以系统地定义氨基酸取代对聚合酶功能的影响； （iii）一种新型的波动测试提供了每种核苷酸取代类别突变率的精确测量； （iv）分子动态建模和生物化学的组合可以定义聚合酶异三聚体中功能上重要的相互作用。 IAV聚合酶的详细分析将以三个目的完成。 （目标1）在50年的H3N2进化中定义了复制速度和忠诚度之间的权衡。竞争测定和波动测试将用于确定PB1自然演化中出现的适应性突变的功能影响。 （目标2）测量所有氨基酸突变在流感病毒RDRP中的结构和功能影响。在不存在和存在诱变核苷的情况下，PB1蛋白的DMS将用于评估每个突变对病毒复制和突变速率的影响。 （AIM 3）定义聚合酶复合物中的氨基酸相互作用如何影响复制和忠诚度。动态建模和体外测定法将用于机械地询问PB2，PB1和PA中的共选择突变如何确定突变率。这项工作具有创新性，因为它独特地结合了一系列关于病毒聚合酶进化的新假设的补充方法。拟议的研究很重要，因为它将定义选择复制率和忠诚度如何塑造流感病毒的短期和长期演变。