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 activity.

Tumor necrosis factor alpha (TNF-α) has an antiviral role in some infections but in dengue virus (DENV) infection it is linked to severe pathology. We have previously shown that TNF-α stimulation cannot activate nuclear factor κB (NF-κB) to the fullest extent in DENV-2-infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-α, focussing particularly on cell death and pro-survival signals. TNF-α stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF-α induced comparable degradation of the inhibitor of NF-κB alpha (IκB-α) and NF-κB activation in mock-infected and DENV-2-infected cells early in infection, later in infection and coinciding with TNF-α-induced cell death, TNF-α-stimulated IκB-α degradation and NF-κB activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells; SphK1 being a known mediator of TNF-α-stimulated survival signals. Transfection experiments demonstrated inhibition of TNF-α-stimulated NF-κB activation by expression of DENV-2 capsid (CA) but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF-α-stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF-α-stimulated survival pathways and show enhanced susceptibility to TNF-α-stimulated cell death, suggesting a role for TNF-α in the killing of healthy productively DENV-2-infected cells. Additionally, the altered ability of TNF-α to activate NF-κB as infection progresses is reflected by the opposing actions of DENV-2 CA and NS5 proteins on TNF-α-stimulated NF-κB activation and could have important consequences for NF-κB-driven release of inflammatory cytokines.

Reduced half-life of holocarboxylase synthetase from patients with severe multiple carboxylase deficiency.

Multiple carboxylase deficiency is a clinical condition caused by defects in the enzymes involved in biotin metabolism, holocarboxylase synthetase (HLCS) or biotinidase. HLCS deficiency is a potentially fatal condition if left untreated, although the majority of patients respond to oral supplementation of 10-20 mg/day of biotin. Patients who display incomplete responsiveness to this therapy have a poor long-term prognosis. Here we investigated cell lines from two such HLCS-deficient patients homozygous for the c.647T>G p.L216R allele. Growth of the patients' fibroblasts was compromised compared with normal fibroblasts. Also the patient cells were not sensitive to biotin-depletion from the media, and growth rates could not be restored by re-administration of biotin. The molecular basis for the HLCS deficiency was further investigated by characterisation of the p.L216R protein. The HLCS mRNA was detected in MCD and normal cell lines. However, protein and enzyme activity could not be detected in the patients' cells. In vitro kinetic analysis revealed that enzyme activity was severely compromised for recombinantly expressed p.L216R and could not be increased by additional biotin. Furthermore, the turn-over rate for the mutant protein was double that of wildtype HLCS. These results help provide a molecular explanation for the incomplete biotin-responsiveness of this p.L216R form of HLCS.