17: Bifunctional role of interferon inducible IFI16 protein inside and outside of endothelial cells

Abstract

The nuclear pathogenic DNA sensor IFI16, induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. As recently demonstrated by our group, it acts as a viral restriction factor against HCMV replication by down-regulating viral early and late but not immediate-early mRNAs as well as their protein expression. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. Our recent results indicate a unique feature displayed by HCMV to overcome the restriction of IFI16, by its upregulation and further mislocalization into egressing virions during the early stages of HCMV infection. IFI16 is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. With our latest experiments using an in-house capture ELISA, we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that free rIFI16 protein severely limits tubulogenesis and transwell migration activities of endothelial cells, while these inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. Furthermore, our in vivo results show that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells, whose characterization is currently in progress. Altogether, our data demonstrate that IFI16 inside a cell can act as viral restriction factor, which can be evaded by herpes viruses, while outside the cell IFI16 can exist as circulating protein in the sera of autoimmune patients which bind endothelial cells causing damage, suggesting a new inflammatory and alarmin function. Proteomic analysis is being performed to characterize the intracellular signaling triggered by extracellular IFI16 and dissect the molecular mechanisms behind its inflammatory role.