Influenza is a contagious respiratory disease in humans, primarily caused by influenza A and B viruses. The severity of pandemic influenza is influenced by several factors, including the virus's ability to evade immune defenses such as autophagy, a key catabolic process within cells. Influenza A virus (IAV) exploits the autophagic pathway to facilitate its replication. In this study, we explored the impact of specific purified honey-derived peptide (H-P) on IAV-induced autophagy signaling, aiming to disrupt viral replication through host-directed strategies and potentially reduce the risk of viral mutation and resistance, as seen with antiviral drugs like Tamiflu. We examined the antiviral potential of H-P in human lung epithelial cells (A549) and compared its effects with Tamiflu and phosphate-buffered saline (PBS) as controls. To assess the safety of H-P, we first evaluated cell viability, lactate dehydrogenase (LDH) release, cell morphology, and cell count following treatment with various concentrations of H-P or Tamiflu in the absence of infection. At lower concentrations, including 5 µM, both treatments showed minimal impact on cell viability, LDH levels, morphology, and cell count. Upon infection with IAV, treatment with 5 µM H-P significantly reduced the expression of both viral nonstructural protein 1 (NS1) and nucleoprotein (NP), whereas Tamiflu treatment at the same concentration reduced only NP expression. Mechanistic analyses revealed that H-P induced both early and late apoptotic signaling in infected cells, as demonstrated by Annexin V staining, suggesting its role in promoting apoptosis during early stages of infection. Additionally, H-P treatment significantly suppressed the expression of autophagy-related genes Atg5 and LC3B during the early stages of infection. It also inhibited the conversion of cytosolic LC3-I to membrane-bound LC3-II, even when autophagy was induced by rapamycin independently of infection. Notably, unlike Tamiflu, H-P increased the production of immune mediators, interferon-beta (IFN-β) and interleukin-6 (IL-6) specifically in response to IAV infection, without altering their expression in the absence of infection. Collectively, these findings highlight the antiviral potential of the naturally derived H-P polypeptide through suppression of IAV-stimulated autophagy, promotion of early apoptosis, and enhancement of antiviral cytokine responses. This suggests a promising host-targeted approach to influenza treatment that may complement or offer alternatives to conventional antiviral therapies like Tamiflu.
