Research Article: Identification of Tyrosine-9 of MAVS as Critical Target for Inducible Phosphorylation That Determines Activation

Date Published: July 26, 2012

Publisher: Public Library of Science

Author(s): Chaoyang Wen, Zhifeng Yan, Xiaoli Yang, Kai Guan, Changzhi Xu, Ting Song, Zirui Zheng, Wenjun Wang, Ying Wang, Man Zhao, Yanhong Zhang, Tao Xu, Jianping Dou, Jingmei Liu, Quanbin Xu, Xiang He, Congwen Wei, Hui Zhong, Naglaa H. Shoukry. http://doi.org/10.1371/journal.pone.0041687

Abstract

Innate immunity to viruses involves receptors such as RIG-I, which senses viral RNA and triggers an IFN-β signaling pathway involving the outer mitochondrial membrane protein MAVS. However, the functional status of MAVS phosphorylation remains elusive.

Here we demonstrate for the first time that MAVS undergoes extensive tyrosine phosphorylation upon viral infection, indicating that MAVS phosphorylation might play an important role in MAVS function. A tyrosine-scanning mutational analysis revealed that MAVS tyrosine-9 (Y9) is a phosphorylation site that is required for IFN-β signaling. Indeed, MAVS Y9F mutation severely impaired TRAF3/TRAF6 recruitment and displayed decreased tyrosine phosphorylation in response to VSV infection compared to wild type MAVS. Functionally, MAVS Y9 phosphorylation contributed to MAVS antiviral function without interfering with its apoptosis property.

These experiments identify a novel residue of MAVS that is crucially involved in the recruitment of TRAF3/TRAF6 and in downstream propagation of MAVS signaling.

Partial Text

The innate immune system, which is characterized by the production of type-1 interferon (IFN)-α/β cytokines and by the activation of natural killer (NK) cells, plays important roles in invading pathogens detection and elimination [1], [2]. In most cell types, RIG-I (retinoic acid inducible gene I) and MDA-5 (melanoma differentiation-associated gene 5) are thought to be the primary receptors that transduce signals using a common intermediary protein called MAVS (mitochondrial antiviral signaling; also known as IPS-1/VISA/Cardif) to stimulate IFN-α/β production in response to infections by RNA viruses [3], [4], [5], [6]. Both RIG-I and MDA5 contain caspase activation and recruitment domains (CARDs) that interact with the CARD domain-containing protein MAVS upon binding to uncapped dsRNA. Binding of RIG-I and MDA-5 to MAVS through CARD-CARD homotypic interactions leads in some way to the stabilization and accumulation of MAVS homodimers. These homodimers are able to directly recruit ubiquitin ligase TRAF3 (tumor necrosis factor receptor-associated factor 3) and to activate its E3 ligase activity. Active TRAF3, in turn, may mediate the recruitment and activation of TBK1 through scaffold molecules, such as NEMO (NF-κB essential modulator) and TANK (TRAF family member-associated NF-κB activator) [7], [8].

Ubiquitination and phosphorylation have emerged as key posttranslational modifications that control induction and shutdown of the interferon response [16], [18], [19]. The mechanism of activation of IKKε and TBK-1 by MAVS following RIG-I engagement involves formation of one or more multimeric complexes in which MAVS potentially interacts with multiple adapter molecules, such as TRAF2/3/6, TRADD (TNFRSF1A-associated via death domain), FADD (Fas -associated via death domain), NEMO, RIP-1 (receptor-interacting serine-threonine kinase 1), TANK (TRAF family member-associated NF-κB activator) and STING (stimulator of interferon genes) [5], [20], [21], [22]. Recent studies showed that ubiquitination at MAVS K500 mediates recruitment of IKKε to MAVS [5], [20], [21], [22]. Interestingly, IKKε recruitment to MAVS resulted in MAVS phosphorylation and negative regulation of the NF-κB pathway with a concomitant decrease in IFN-β and ISG (interferon stimulated gene) expressions [23]. However, the exact functions of MAVS tyrosine phosphorylation that regulate the IFN-β response are incompletely understood. In the present study, we demonstrated for the first time that MAVS undergoes extensive tyrosine phosphorylation following viral infection. More specifically, tyrosine-9 of MAVS was identified as a phosphorylation site that is required for IFN-β signaling. Indeed, MAVS Y9F mutation directly reduced TRAF3 and TRAF6 recruitment. Functionally, MAVS tyrosine-9 phosphorylation contributed to MAVS antiviral function without interfering with its apoptosis property.

Source:

http://doi.org/10.1371/journal.pone.0041687