NEMO Binds Ubiquitinated TANK-Binding Kinase 1 (TBK1) to Regulate Innate Immune Responses to RNA Viruses

Lingyan Wang,Martin E Dorf,Shitao Li,Eliane F Meurs

doi:10.1371/journal.pone.0043756

Abstract

RIG-I-like receptors (RLR) are intracellular sensors utilized by nearly all cell types for recognition of viral RNA, initiation of antiviral defense, and induction of type I interferons (IFN). TBK1 is a critical kinase implicated in RLR-dependent IFN transcription. Posttranslational modification of TBK1 by K63-linked ubiquitin is required for RLR driven signaling. However, the TBK1 ubiquitin acceptor sites and the function of ubiquitinated TBK1 in the signaling cascade are unknown. We now show that TBK1 is ubiquitinated on residues K69, K154, and K372 in response to infection with RNA virus. The K69 and K154 residues are critical for innate antiviral responses and IFN production. Ubiquitinated TBK1 recruits the downstream adaptor NEMO through ubiquitin binding domains. The assembly of the NEMO/TBK1 complex on the mitochondrial protein MAVS leads to activation of TBK1 kinase activity and phosphorylation of the transcription factor, interferon response factor 3. The combined results refine current views of RLR signaling, define the role of TBK1 polyubiquitination, and detail the mechanisms involved in signalosome assembly.

Highlights

An important aspect of host resistance against viral infections is the production of type I interferons (IFN)
Overexpression of TANK-Binding Kinase 1 (TBK1)(N385) induced low but significant levels of luciferase reporter activity driven by the interferon-stimulated response element (ISRE) which requires activation by interferon regulatory factor 3 (IRF3) (Fig. 1C)
To examine the biologic properties associated with these sites, HEK293 cells were transfected with TBK1 KRR mutants along with K63-only ubiquitin

Summary

Introduction

An important aspect of host resistance against viral infections is the production of type I interferons (IFN). Cytosolic receptors, such as the RIG-I like receptors (RLR), sense viral RNA in most cell types. Following RNA recognition, RLRs translocate onto a scaffold molecule termed MAVS which serves as a platform for coordinating downstream innate immune signaling [1,2]. RLR engagement of MAVS leads to activation of downstream kinases and transcription factors, including TBK1 and interferon regulatory factor 3 (IRF3), respectively. Following RLR-MAVS interaction, TBK1, a constitutively and ubiquitously expressed serinethreonine kinase, catalyzes phosphorylation of IRF3 [3,4,5,6]. The mechanisms by which RLR signals recruit and activate TBK1 are not well understood

Methods

Results

Conclusion