A33-05 Activation of α7 Nachr Promotes Influenza A Virus Replication in Lung Epithelial Cells via the Camk2β/nedd4/ifitm3 Pathway

Rationale The α7 nicotinic acetylcholine receptor (α7 nAChR) is expressed on lung epithelial cells and is a key mediator of the cholinergic anti-inflammatory pathway. Our previous work established that activating α7 nAChR enhances influenza A virus (IAV) replication via the NEDD4L-ASK1-p38MAPK pathway (J Med Virol, 2024). Building on this, we now seek to determine if an alternative pathway—involving CAMK2β, NEDD4, and IFITM3—mediates the pro-viral effects of α7 nAChR activation in alveolar epithelial cells. Methods Human alveolar type II epithelial cells (A549) were pretreated with the specific α7 nAChR agonist GTS-21 and subsequently infected with IAV (strain PR8). Viral replication was assessed by quantifying PR8 M gene copies (qPCR) and NP/NS1 protein levels (western blot). To investigate the mechanism, we generated A549 cells with stable knockdown or overexpression of CAMK2B or NEDD4 using lentiviral constructs. These engineered cells were used to test the involvement of CAMK2β and NEDD4 in the GTS-21-mediated effect on IAV replication. Results Activation of α7 nAChR with GTS-21 significantly promoted IAV replication and concurrently downregulated the expression of the antiviral restriction factor IFITM3. This activation also reduced the expression of Ca2+/calmodulin-dependent protein kinase 2β (CAMK2β). Functional studies confirmed that CAMK2B overexpression enhanced IFITM3 expression, while its knockdown suppressed the IFNβ/STAT1 signaling axis, reduced IFITM3 levels, and facilitated viral replication. Furthermore, α7 nAChR activation enhanced the expression of the E3 ubiquitin ligase NEDD4, an effect potentiated by CAMK2B knockdown during IAV infection. Critically, knockdown of NEDD4 reduced viral titers and abolished the pro-viral effect of GTS-21. Conclusion Our findings demonstrate that activation of α7 nAChR promotes IAV replication in lung epithelial cells. This effect is mediated through a novel signaling pathway involving the downregulation of CAMK2β, subsequent upregulation of NEDD4, and the resulting suppression of the antiviral protein IFITM3. This study identifies the α7 nAChR/CAMK2β/NEDD4 axis as a potential target for host-directed therapies against influenza.