Research Article: Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

Date Published: January 23, 2014

Publisher: Public Library of Science

Author(s): Domingo Miranzo-Navarro, Katharine E. Magor, Pierre Boudinot.

http://doi.org/10.1371/journal.pone.0086968

Abstract

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25 activation of duck RIG-I.

Partial Text

RIG-I is an intracellular detector of 5′ triphosphate RNA that activates a signaling pathway leading to the production of type I interferon and initiation of the antiviral state [1]. The three dimensional structures of RIG-I from several species [2], [3], [4], [5] provide a molecular model for RIG-I activation [6]. The pathway starts upon sensing of viral RNA by the RIG-I helicase and regulatory domains, which undergo a conformational change, acting as a molecular camshaft that uses energy from ATP hydrolysis to expose the two caspase activator recruitment domains (CARDs) to the cytoplasm [7]. Activated CARD domains of RIG-I interact with CARD domains of MAVS (VISA, CARDIF, IPS-I) [8], [9], [10], which aggregate in a prion-like structure [11]. This conformational change allows MAVS to serve as a platform to recruit the other components of the pathway in a multiprotein signaling complex [12]. Downstream, activation of IRF3 and NF-κβ transcription factors induce type I interferon and production of proinflamatory cytokines [13].

Here we examine regulation of RIG-I in the natural host of avian influenza. Human RIG-I is activated by K63-linked polyubiquitin attached at lysine 172 in the second CARD domain [17], or interaction with unanchored K63-linked polyubiquitin chains, also involving lysine 172 [19], a residue that is missing in ducks and other birds. We demonstrate that chicken and duck TRIM25 are functional, and duck CARD domains are ubiquitinated. The duck TRIM25-RIG-I interaction plays a role in the activation of IFN-β production as demonstrated by the nonfunctional splice variant of RIG-I, which is unable to interact with TRIM25, and does not induce IFN-β. Using mass spectrometry to analyze ubiquitinated CARD domains, we determined that K167 and K193 were the ubiquitination sites of duck CARD domains. We showed that mutation of each site independently did not disrupt ubiquitination, however a double mutant with both K167R and K193R, was unable to undergo ubiquitination. Surprisingly, this double mutant retained activity and the ability to be activated by duck TRIM25. Thus, duck TRIM25 can activate RIG-I through a process that does not depend on attachment of ubiquitin chains to the CARD domains of RIG-I.

 

Source:

http://doi.org/10.1371/journal.pone.0086968