Abstract:
Introduction: Human Adenoviruses (HAdVs) are obligate intracellular organisms that typically subvert epithelial cells by converting them into virion-producing factories. HAdVs encode a protein within the E3 region, termed adenovirus death protein (ADP), which facilitates cell lysis at the end of the viral cycle, thereby enhancing the dissemination of progeny virions via an unknown mechanism. Sphingolipids, a diverse pool of structural and bioactive lipids, are known mediators of various cellular pathways. One class of sphingolipids, ceramides, have an established role in the apoptotic cellular response to stress and were shown to be synthesized de novo shortly prior to cell lysis induced by wild type adenoviral infection. However, in cells infected with the E3-deleted mutant counterpart (dl7001), total ceramide, as measured by the diacylglycerol kinase assay, accumulated similarly but cell lysis did not occur due to the lack of ADP. Our current aim is to investigate the dynamics of sphingolipid changes induced by wild- type HAdV (rec700) and its E3-deleted mutant counterpart (dl7001) infection of A549 human lung adenocarcinoma cells by using Liquid Chromatography Tandem Mass Spectrometry (LC- MS) in order to understand the distinct cellular responses to these two viruses.
Methods: Cell viability was assessed by trypan dye exclusion. Analysis of cellular and viral gene expression was performed by real time PCR. LC-MS was performed on mock-infected cells, in addition to cells infected with either rec700 or dl7001. Western blotting was used to assess the expression levels of dihydroceramide desaturase.
Results: Differential patterns of sphingolipid biosynthesis were observed in rec700- and dl7001- infected A549 cells. Whereas dihydroceramides accumulated significantly only in rec700- infected cells, ceramides accumulated significantly only in dl7001-infected cells.
Conclusion: We speculate that an E3 protein is interfering in the final step of the de novo synthesis pathway undertaken by the enzyme that converts dihydroceramide into ceramide.