Newcastle disease virus induces clathrin-mediated endocytosis to establish infection through the activation of PI3K/AKT signaling pathway by VEGFR2

ABSTRACT The phosphatidyl-inositol 3-kinase/serine-threonine kinase (PI3K/ AKT) signaling pathway constitutes a classical phosphorylation cascade that integrates tyrosine, lipid, and serine acid-threonine phosphorylation, affecting cell function. The pathway is vulnerable to viral infection. Newcastle disease virus (NDV) poses a significant threat to the global poultry industry; however, its mechanism of early viral cell invasion and pathogenesis remain unclear. Previous in vivo and in vitro studies have shown that NDV infection activates PI3K/AKT signaling; however, it remains unclear whether NDV establishes infection through endocytosis regulated by this pathway. This study aimed to examine whether different genotypes of NDV strains could activate the PI3K/AKT signaling pathway within 2 h of in vitro infection. This activation, which relies on PI3K phosphorylation, remains unaffected by the phosphorylation-phosphatase and tensin homolog/phosphatase and tensin homolog (p-PTEN/PTEN) signaling pathway. Moreover, inhibition of PI3K activity impedes NDV replication. Additionally, interfering with the PI3K regulatory subunit p85 has no significant effect on NDV replication. Conversely, the tyrosine kinase activity upstream of PI3K can influence AKT activation and viral replication, particularly through vascular endothelial growth factor receptor 2 (VEGFR2). Additionally, NDV F protein primarily mediates PI3K and AKT phosphorylation to activate the PI3K/AKT signaling pathway. NDV F and VEGFR2 proteins, along with the PI3K p85α subunit, interact and co-localize at the cell membrane. NDV-induced PI3K/AKT signaling pathway activation impacts clathrin-mediated endocytosis, with VEGFR2 playing a pivotal role. In conclusion, this study shows that NDV infection is established early through F protein binding to VEGFR2, activating the PI3K/AKT signaling pathway and inducing clathrin-mediated endocytosis, supporting infection prevention and control measures. IMPORTANCE Newcastle disease virus (NDV) is a threat to the global poultry industry; however, the mechanisms of NDV infection remain unclear. NDV affects the phosphatidyl-inositol 3-kinase/serine-threonine kinase (PI3K/ AKT) signaling pathway, requiring endocytosis for successful infection. Based on previous studies, we identified a close correlation between NDV infection and replication and the PI3K/AKT signaling pathway activity. This study examined the molecular mechanisms through which NDV activates the PI3K/AKT signaling pathway to regulate endocytosis and facilitate infection. This study showed that early-stage in vitro NDV infection activated the PI3K/AKT signaling pathway, enhancing clathrin-mediated endocytosis, crucial for infection onset. Notably, this process involves the interaction between NDV F protein and the vascular endothelial growth factor receptor 2 tyrosine kinase, leading to the subsequent binding and phosphorylation of the PI3K p85α regulatory subunit. This activation primes PI3K, initiating a cascade that promotes clathrin-mediated endocytosis. Our findings elucidate how NDV capitalizes on the PI3K/AKT signaling pathway to establish infection through endocytosis.