High throughput profiling identified PA-L106R amino acid substitution in A(H1N1)pdm09 influenza virus that confers reduced susceptibility to baloxavir in vitro

• Published in: Antiviral research Vol. 229; p. 105961
• Main Authors: Chen, Dongdong, Su, Wen, Choy, Ka-Tim, Chu, Yan Sing, Lin, Chi Ho, Yen, Hui-Ling
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2024
• Subjects: Amino Acid Substitution; Animals; Antiviral Agents - pharmacology; Baloxavir; Cap-dependent endonuclease inhibitor; Deep mutational scanning; Dibenzothiepins - pharmacology; Dogs; Drug Resistance, Viral - genetics; Fitness; High-Throughput Nucleotide Sequencing; Humans; Influenza A Virus, H1N1 Subtype - drug effects; Influenza A Virus, H1N1 Subtype - genetics; Influenza virus; Influenza, Human - drug therapy; Influenza, Human - virology; Madin Darby Canine Kidney Cells; Morpholines - pharmacology; Oxazines - pharmacology; Pyridones - pharmacology; Reduced susceptibility; RNA-Dependent RNA Polymerase - genetics; Thiepins - pharmacology; Triazines - pharmacology; Viral Proteins - genetics; Virus Replication - drug effects; Reduced susceptibility; Cap-dependent endonuclease inhibitor; Deep mutational scanning; Influenza virus; Baloxavir; Fitness
• ISSN: 0166-3542; 1872-9096; 1872-9096
• DOI: 10.1016/j.antiviral.2024.105961

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. [Display omitted] •Deep mutation scanning of A (H1N1)pdm09 PA endonuclease domain was performed under increasing BXA concentrations in vitro.•The I38 T/M substitutions associated with reduced BXA susceptibility emerged within 5 passages from independent replicates.•L to R change at a conserved PA residue 106 conferred >10-fold reduced susceptibility to BXA with moderate fitness loss.