MEK/ERK signaling and Bcl-2 family protein changes in influenza-infected cells with differing sulfatide levels.

Influenza A virus (IAV) exploits host pathways to maximize progeny production. Sulfatide, a sulfated glycosphingolipid on the plasma membrane, has been implicated in enhanced influenza replication; however, the underlying host factors remain unclear. Using sulfatide-low COS7 cells, sulfatide-enriched SulCOS1 cells, and Madin-Darby canine kidney (MDCK) cells that endogenously express sulfatide, we observed higher infectious viral yields in MDCK and SulCOS1 cells than in COS7 cells, without a corresponding increase in the number of initially infected cells. IAV infection triggered time-dependent activation of mitogen-activated protein kinase (MAPK) signaling in sulfatide-enriched cells, with a prominent response in extracellular signal-regulated kinases 1 and 2 (ERK1/2). Treatment with U0126, a mitogen-activated protein kinase kinase (MEK)/ERK-pathway inhibitor, reduced viral output without a marked increase in lactate dehydrogenase (LDH) release, although ERK dependence remains unresolved. To assess mitochondrial apoptosis-related responses, we examined the pro-apoptotic BH3-only protein Bim. In sulfatide-enriched SulCOS1 cells, Bim abundance decreased during the late phase of infection, whereas Bim remained relatively stable in COS7 cells. In MDCK cells, U0126 tended to preserve Bim abundance after infection. Although caspase-3 was activated after infection, pan-caspase inhibition did not detectably reduce hemagglutination (HA) titers under the tested conditions. In addition, changes in Bcl-2 family protein abundance were observed in MDCK and SulCOS1 cells, and Bax-targeting peptides increased influenza A and B virus production in MDCK cells. Together, these data support a model in which sulfatide-associated MEK/ERK signaling, Bim-related mitochondrial checkpoint changes, and other downstream processes may collectively contribute to efficient influenza virus production.