Viperin interacts with the kinase IRAK1 and the E3 ubiquitin ligase TRAF6, coupling innate immune signaling to antiviral ribonucleotide synthesis

Abstract

Virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) is a radical SAM enzyme that plays a multifaceted role in the cellular antiviral response. Viperin has recently been shown to catalyze the SAM-dependent formation of 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), which inhibits some viral RNA polymerases. Viperin is also implicated in regulating Lys-63-linked polyubiquitination of interleukin-1 receptor-associated kinase-1 (IRAK1) by the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) as part of the Toll-like receptor-7 and -9 (TLR7/9) innate immune signaling pathways. In these pathways, the poly-ubiquitination of IRAK1 by TRAF6 is necessary to activate IRAK1, which then phosphorylates downstream targets and ultimately leads to the production of type I interferons. That viperin is a component of these pathways suggested that its enzymatic activity might be regulated by interactions with partner proteins. To test this idea, we have reconstituted the interactions between viperin, IRAK1, and TRAF6 by transiently expressing these enzymes in HEK 293T cells. We show that IRAK1 and TRAF6 increase viperin activity ∼10-fold to efficiently catalyze the radical-mediated dehydration of CTP to ddhCTP. Furthermore, we found that TRAF6-mediated ubiquitination of IRAK1 requires the association of viperin with both IRAK1 and TRAF6. Ubiquitination appears to depend on structural changes in viperin induced by SAM binding, but, significantly, does not require catalytically active viperin. We conclude that the synergistic activation of viperin and IRAK1 provides a mechanism that couples innate immune signaling with the production of the antiviral nucleotide ddhCTP.

Keywords: S-adenosylmethionine domain containing 2 (RSAD2); TNF receptor-associated factor 6 (TRAF6); Toll-like receptor (TLR); antiviral protein; innate immunity; interleukin 1 receptor associated kinase 1 (IRAK1); protein kinase; radical SAM enzymes; signal transduction; ubiquitin ligase.