Abstract：
Baloxavir acid (BXA) is a pan-influenza antiviral that targets the cap-dependent endonuclease of the polymerase acidic (PA) protein required for viral mRNA synthesis. To gain a comprehensive understanding on the molecular changes associated with reduced susceptibility to BXA and their fitness profile, we performed a deep mutational scanning at the PA endonuclease domain of an A (H1N1)pdm09 virus. The recombinant virus libraries were serially passaged in vitro under increasing concentrations of BXA followed by next-generation sequencing to monitor PA amino acid substitutions with increased detection frequencies. Enriched PA amino acid changes were each introduced into a recombinant A (H1N1)pdm09 virus to validate their effect on BXA susceptibility and viral replication fitness in vitro. The I38 T/M substitutions known to confer reduced susceptibility to BXA were invariably detected from recombinant virus libraries within 5 serial passages. In addition, we identified a novel L106R substitution that emerged in the third passage and conferred greater than 10-fold reduced susceptibility to BXA. PA-L106 is highly conserved among seasonal influenza A and B viruses. Compared to the wild-type virus, the L106R substitution resulted in reduced polymerase activity and a minor reduction of the peak viral load, suggesting the amino acid change may result in moderate fitness loss. Our results support the use of deep mutational scanning as a practical tool to elucidate genotype-phenotype relationships, including mapping amino acid substitutions with reduced susceptibility to antivirals.
摘要：
Baloxavir酸（BXA）是一种泛流感抗病毒剂，其靶向病毒mRNA合成所需的聚合酶酸性（PA）蛋白的帽依赖性内切核酸酶。为了全面了解与BXA易感性降低相关的分子变化及其适应度，我们对A(H1N1)pdm09病毒的PA核酸内切酶结构域进行了深度突变扫描.在增加浓度的BXA下体外连续传代重组病毒库,随后进行下一代测序以监测检测频率增加的PA氨基酸取代。将富集的PA氨基酸变化各自引入到重组A(H1N1)pdm09病毒中,以验证它们在体外对BXA易感性和病毒复制适应性的影响。已知赋予对BXA敏感性降低的I38T/M取代总是在5个连续传代内从重组病毒库中检测到。此外,我们发现了一个新的L106R取代,它出现在第3代,并使BXA的易感性降低了10倍以上.PA-L106在季节性甲型和乙型流感病毒中高度保守。与野生型病毒相比，L106R取代导致聚合酶活性降低和峰值病毒载量的轻微降低，这表明氨基酸的变化可能会导致适度的健身损失。我们的结果支持使用深度突变扫描作为阐明基因型-表型关系的实用工具。包括定位氨基酸取代降低对抗病毒药物的敏感性。
