Kaposi's sarcoma herpesvirus viral FLICE inhibitory protein modulates A20 deubiquitinase activity

Abstract

KSHV viral FLICE inhibitory protein (vFLIP) is a potent activator of NF-κB signalling and an inhibitor of apoptosis and autophagy. Inhibition of vFLIP function and NF-κB signalling promotes lytic reactivation. Here we provide evidence for a novel function of vFLIP through inhibition of the deubiquitinating (DUB) activity of the negative regulator, A20. We demonstrate direct interaction of vFLIP with Itch and A20 and provide evidence for subsequent loss of A20 DUB activity. Our results provide further insight into the function of vFLIP in the regulation of NF-κB signalling.

Keywords: CASP8; FADD-like apoptosis regulating protein; Kaposi's sarcoma-associated herpesvirus; deubiquitinating enzymes; herpesvirus 8; human; ubiquitin.

Fig. 1.. vFLIP interacts with Itch and A20. (a) vFLIP interacts with Itch. (b) vFLIP interacts with A20. For both experiments 293 T cells were transfected with Flag–Itch, Flag–A20, and/or myc–vFLIP, where indicated. Empty vector was used to maintain equal amounts of DNA. Itch or A20 were immunoprecipitated with Flag antibody-conjugated agarose beads and immunoprecipitates were analysed by immunoblot against Flag and myc. Data shown are representative of a minimum of three replicates.

Fig. 2.. vFLIP modulates A20 DUB activity. (a) vFLIP inhibits A20 DUB activity in vitro. In 20 µl reactions the following reagents were added where indicated: tetra-K63 Ub (Boston Biochem) (500 nM), V5-vFLIP (1,5,10 µM), and A20 (2 µM). Reactions were incubated at 37 °C for 2 h following addition of 4× Laemmli loading buffer. Samples were run on 15 % SDS-PAGE gel and analysed via immunoblot with antibody against ubiquitin. (b) vFLIP abrogates the deubiquitination of RIPK1. HEK-293T cells were transfected with either empty vector control, HA-tagged WT Ub, and myc-tagged vFLIP where indicated, and 24 h post-transfection cells were treated with TNFA at 0, 0.5, and 2 h before harvesting, and analysed via immunoprecipitation with anti-RIPK1. Following immunoprecipitation, lysates were analysed by immunoblotting with anti-HA and anti-RIPK1. Data shown are representative of a minimum of three replicates.