Thesis & Dissertations

Effect of Acid Sphingomyelinase on Propagation of Influenza A Virus (IAV‎)


Background: Influenza A virus (IAV) is a major human respiratory pathogen causing annual epidemics as well as periodic pandemics. Although many studies were done to investigate the signaling pathways induced during IAV infection, the signaling events triggered by IAV that mediate its internalization and initiation of the infection cycle are still not fully understood. Lipid rafts, membrane microdomains enriched in sphingomyelin (SM) and cholesterol, have been shown to be important for IAV infection. Sphingomyelinases (SMases) are enzymes that catalyze the hydrolysis of SM into phosphorylcholine and ceramide. Attachment of some pathogens to their receptors at the plasma membrane can trigger the activation of acid sphingomyelinase (ASMase) and consequently the formation of ceramide-enriched membrane platforms, which are required for pathogen uptake. Although it has been previously shown that IAV utilizes lipid rafts during its entry and budding from the cells, the role of SMases in IAV infection has not been addressed yet. Understanding the role of ASMase during IAV infection is important to identify novel host targets for developing new influenza antivirals Aim: To better understand the role of SM pathway during IAV infection, and to investigate if ASMase modulates IAV replication in lung epithelial cells. Methods: The effect of desipramine, an ASMase inhibitor, on replication of IAV in human lung adenocarcinoma epithelial cell line (A549) was investigated. IAV replication in ASMase deficient fibroblasts (Neimann-Pick Disease cells or NPD) and normal human fibroblasts was assessed. ASMase activity was assayed in mock-infected and IAV-infected cells at different time points. Additionally, the effect of SM depletion using exogenous ASMase on IAV replication was examined. Viral titers were determined by either TCID50 or plaque assay. Results: Our results show that pharmacological inhibition of ASMase by desipramine had no effect on IAV replication in A549 cells. On the other hand, IAV replication in NPD cells was not compromised, but was rather more efficient compared to normal fibroblasts. In addition, ASMase activity levels were not changed in presence or absence of IAV infection. Depletion of plasma membrane SM by exogenous ASMase reduced virus production in A549 cells, indicating that SM present in the plasma membrane might be required for IAV infection. Conclusion: Our study reveals that ASMase negatively modulates IAV infection and that SM is an important modulator that is required for IAV replication. Studies to understand the exact mechanism underlying the modulation of IAV replication by SM are ongoing. Further, investigations of the role of other sphingolipid pathways during IAV replication are underway.


Amani Walid Audi


Jamila Borjac, Hassan Mohammad Zaraket