Atractylenolide-Ⅲ binds non-structural protein-1 to suppress influenza A by modulating macrophage polarization and alternative polyadenylation

Le résumé fourni par la source

• Atractylenolides (ACTs, including ACT-Ⅰ, -Ⅱ, -Ⅲ) specifically bind with the influenza A virus (IAV) non-structural protein 1 (NS1). • ACT-Ⅲ shows anti-influenza activity both in vitro and in vivo , effectively mitigating virus-induced lung injury. • By binding to NS1, ACT-Ⅲ modulates IAV infection-associated pro-inflammatory signaling pathways. • ACT-Ⅲ regulates the alternative polyadenylation (APA)of host mRNA by disrupting NS1-cleavage and polyadenylation-specific factor 4 (CPSF4) interaction. Drug-resistant influenza demands novel antiviral treatments. Non-structural protein 1 (NS1) of influenza A virus (IAV) regulates the viral life cycle and host immune response, thus becoming a promising therapeutic target. The atractylenolide (ACT) -Ⅲ exhibits notable anti-IAV efficacy; however, its in vivo anti-IAV activity and the underlying mechanisms need further exploration. We explored the binding affinity of ACT-Ⅲ with NS1 and elucidated the in vivo anti-influenza activity and underlying mechanism of ACT-Ⅲ. Surface plasmon resonance (SPR) analysis was utilized to determine the binding affinity of ACT-Ⅲ to NS1. The anti-IAV activity of ACT-Ⅲ was further tested in vitro using IAV-infected lung epithelial cells and in vivo in IAV-challenged mice . The evaluation criteria included the assessment of virus-associated cytopathies, viral protein and gene expression, virus titer, mice weight loss, survival curve, and lung pathology . Transcriptomic sequencing was conducted to evaluate the inflammation regulatory function of ACT-Ⅲ in IAV-infected Raw264.7 cells. Quantitative real-time PCR (qRT-PCR) was used to detect the target gene expression. Flow cytometry was utilized to assess macrophages polarization. Co-immunoprecipitation was employed to examine the impact of ACT-Ⅲ on the interaction between NS1 and cleavage and polyadenylation-specific factor 4 (CPSF4). ACT-Ⅲ exhibited anti-IAV activity in vitro and in vivo , mitigating virus-induced lung injury. Mechanistically, ACT-Ⅲ impeded IAV infection and viral induced inflammation by binding to viral NS1, which reduced the activation of pro-inflammatory signaling, and disrupted the NS1-CPSF4 interaction that mediates alternative polyadenylation (APA). ACT-Ⅲ inhibits IAV replication and viral pneumonia by binding to NS1, showing potential as a therapeutic agent for IAV treatment. In macrophages challenged with influenza A virus (IAV), atractylenolide-Ⅲ (ACT-Ⅲ) exerts dual antiviral mechanisms through targeting non-structural protein 1 (NS1). Mechanistically, ACT-Ⅲ not only attenuates pro-inflammatory signaling by modulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) axis and potentiating type I interferons (IFN-Is) induction, but also directly disrupts the NS1-cleavage and polyadenylation specificity factor subunit 4 (CPSF4) interaction through structural interference. This structural disruption effectively blocks the viral protein's ability to hijack the host's alternative polyadenylation (APA) machinery for mRNA processing. Theses actions collectively reduces both viral pathogenicity and IAV-induced dysregulation of macrophage polarization dynamics.

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