Abstract

Protein kinase C alpha (PKCα) is a central signaling molecule implicated in various cellular processes, including viral infections. However, its role in fish viruses, particularly nervous necrosis virus (NNV), remains elusive. Here, we report that PKCα from marine medaka (MmPKCα) facilitates red-spotted grouper NNV (RGNNV) entry by bridging viral receptor marine medaka myosin light chain 3 (MmMYL3, a known RGNNV receptor mediating macropinocytosis) binding to downstream actin dynamics. RGNNV infection upregulates MmPKCα expression and activates its phosphorylation. Gain- and loss-of-function studies demonstrated that MmPKCα enhances NNV entry, dependent on its kinase activity. We found that MmPKCα interacts with the RGNNV capsid protein via its C-terminal (CT) domain. Although MmPKCα localized to the cell surface, it did not function as an RGNNV receptor, evidenced by unaltered viral binding, inability to render non-susceptible cells permissive, and lack of inhibition by anti-PKCα antibodies/recombinant MmPKCα protein. Instead, MmPKCα directly interacts with MmMYL3 and is essential for MmMYL3-mediated macropinocytosis. Furthermore, MmPKCα binds to marine medaka cofilin (MmCFL1/2) through its CT domain and suppresses cofilin phosphorylation at Ser3, thereby activating cofilin and promoting actin rearrangement critical for macropinocytosis. Our study uncovers a novel MmMYL3-MmPKCα-cofilin signaling axis that RGNNV exploits to enter host cells, highlighting PKCα as a potential therapeutic target for controlling NNV infection.