Sphingosine kinase 1 (SK1) is a host cell enzyme that we have previously shown has altered activity in dengue virus (DENV) infected cells. Control of SK1 activity can be mediated by plasma membrane translocation and SK1 activation can be associated with cell survival and signalling pathways; pathways that may be important for providing an active environment for viral replication. In this study we have investigated the requirement for SK1 during DENV infection and the cellular localisation of SK1 in DENV-infected cells.
Methods: Wild-type (WT) and SK1 knock-out (-/-) murine embryonic fibroblasts were DENV or mock infected and infectious virus production quantitated by plaque assay. Additionally, cells were fixed and endogenous and induced SK1, from a FlpIn tet-FLAG-SK1 expression system in HEK-293, was analysed by immunostaining and confocal microscopy.
Results and Conclusions: Infectious virus production was 100X lower from WT compared with SK1-/- MEF suggesting an enhancing role for SK1 in promoting DENV infection. In HEK-293 cells endogenous SK1 was present in a punctate staining pattern primarily in the cell cytoplasm. A similar distribution of induced FLAG-SK1 was observed. Results in particular analysed the localisation of SK1 at at the plasma membrane. Future experiments will aim to analyse the translocation of SK1 to intracellular virus-containing vesicles.