O011 IL-17 receptor adaptor Act1/CIKS plays an evolutionarily conserved role in antiviral signalling

Abstract

Introduction Double stranded RNA-induced antiviral gene expression in mammalian cells depends on activation of Interferon Regulatory Factor 3 (IRF3) [1] , [2] . As the expression of the Interleukin-17 receptor adaptor protein Act1 is induced in response to double-stranded RNA, we asked whether Act1 is involved in the IRF3-dependent antiviral signalling. Methods RNA interference was used to knockdown Act1 expression in primary human fibroblasts to test the protein’s involvement in polyinosinic:polycytidylic acid (poly(I:C))-induced antiviral gene expression, measured by quantitative PCR (qPCR), and IRF3 activation detected with a phospho-IRF3 antibody. Also, transient transfection of human 293 ET cells with expression plasmids, encoding Act1 and other components of antiviral pathways, was used to monitor the activation of IRF3 using an IRF3-specific luciferase-based reporter assay and qPCR. Point mutations and deletions of Act1 were created to map specific regions responsible for its interactions with other components of the antiviral signalling. Zebrafish Act1 was cloned into a mammalian expression vector and compared to its human counterpart in terms of its ability to trigger IRF3 activation (using the above-mentioned reporter assay) and antiviral gene expression measured by qPCR. Results (1) The siRNA-mediated knockdown of Act1 inhibits antiviral gene expression and IRF3 phosphorylation induced by poly(I:C) stimulation, while the overexpression of Act1 potentiates IRF3-driven antiviral gene expression; (2) Act1 interacts with the components of antiviral signalling pathways, IKKe and IRF3, using distinct domains; (3) Act1-induced IRF3 activation can be blocked specifically by co-expression of a catalytically-inactive mutant of IKKe; (4) mutants of IRF3, either lacking the C-terminus or mutated at the key phosphorylation sites, important for its activation by IKKe, do not support Act1-dependent IRF3 activation; (5) Zebrafish Act1 protein is able to trigger antiviral gene expression in human cells. Conclusion Act1 is a novel component of antiviral signalling, which functions as a signalling adaptor of IKKe and IRF3. Comparison of mammalian and fish Act1 proteins suggests evolutionary conservation of the antiviral function of Act1 in vertebrates.