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

Abstract

Background: 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.

Purpose: We explored the binding affinity of ACT-Ⅲ with NS1 and elucidated the in vivo anti-influenza activity and underlying mechanism of ACT-Ⅲ.

Study design and methods: 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).

Results: 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).

Conclusion: ACT-Ⅲ inhibits IAV replication and viral pneumonia by binding to NS1, showing potential as a therapeutic agent for IAV treatment.

Keywords: Alternative polyadenylation; Atractylenolide-ⅲ; Influenza a virus; Macrophage polarization; Non-structural protein-1.