A novel role for neutral sphingomyelinase during Influenza A Virus replication

Abstract

Influenza A virus (IAV) is one of the most common causative agents of acute respiratory tract infections worldwide. It causes significant morbidity and mortality during its seasonal outbreaks and can occasionally cause pandemics. Current influenza antiviral drugs target viral proteins, which can quickly evolve resistance to existing drugs. Therefore, targeting host cell factors during infection is a promising antiviral approach as their genes are less prone for mutation, and thus the selection of resistance. Numerous studies have demonstrated the role of sphingolipids in various aspects of the life cycles of several viruses including attachment and fusion, intracellular transport, replication, and budding. Previous studies conducted in our laboratory have shown that IAV infection leads to increase in cellular ceramide. Knowing that neutral sphingomyelinase (nSMase) is a key player in stress-induced production of ceramide by hydrolyzing the membrane lipid sphingomyelin (SM), we hypothesized that it could play a role during IAV infection. Therefore, the aim of this study was to investigate the role of nSMase during IAV infection in A549 cells, human lung adenocarcinoma epithelial cells. We first investigated if IAV induces ceramide accumulation at the plasma membrane and whether this accumulation is mediated by nSMase. This was addressed by assessing ceramide accumulation in cells infected with IAV in the presence or absence GW4869, a nSMase inhibitor, by using confocal microscopy. In addition, we measured virus replication upon inhibition of nSMase. Viral titers were determined using plaque assay. Further, we assayed the activity of nSMase in IAV-infected lung epithelial cells at different time points using Amplex® Red Sphingomyelinase assay kit. Our results indicate that IAV induces ceramide accumulation at the plasma membrane. Inhibition of nSMase prevented the accumulation of ceramide in infected cells, suggesting that ceramide increase is mediated by nSMase. Moreover, nSMase inhibition reduced IAV repli