Tumour necrosis factor alpha (TNF-alpha) stimulation of cells with established dengue virus type 2 infection induces cell death that is accompanied by a reduced ability of TNF-alpha to activate nuclear factor kappaB and reduced sphingosine kinase-1 a

TNF-alpha has an anti-viral role in some infections but in dengue virus (DENV) infection is linked to severe pathology. We have previously shown that TNF-alpha cannot activate NFkappaB to the full extent in DENV-2 infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-alpha, with particular focus on cell death and pro-survival signals. TNF-alpha stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF-alpha induced comparable IkappaB-alpha degradation and NFkappaB activation in mock-infected and DENV-2-infected cells early in infection, later in infection and co-inciding with TNF-alpha-induced cell death, TNF-alpha-stimulated IkappaB-alpha degradation and NFkappaB activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells, a known mediator of TNF-alpha-stimulated survival signals. Transfection of cells to express DENV-2 CA or NS5 protein demonstrated inhibition of TNF-alpha-stimulated NFkappaB activation by expression of DENV-2 CA but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF-alpha stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF-alpha-stimulated survival pathways and show enhanced susceptibility to TNF-alpha-stimulated cell death, suggesting a role for TNF-alphain clearance of healthy productively DENV-infected cells. Additionally, the altered ability of TNF-alpha to activate NFkappaB as infection progresses is reflected by opposing actions of DENV CA and NS5 proteins on TNF-alpha stimulated NFkappaB activation and could have important consequences for NFkappaB-driven release of inflammatory cytokines.