Abstract
RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses.
Highlights
RIG-I, melanoma differentiation factor 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) belong to the DExD/H-box family of helicases[2]
Our study demonstrates that the hierarchical mechanism of multi-site/ type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in the type I IFN signaling pathway during antiviral immune responses
In contrast to RIG-I-2CARD, the K164R or K172R single mutants of FL-RIG-I only partially reduced ISRE activation (Fig. 1C), whereas FL-RIG-I double mutation with K164R and K172R (DM) significantly blocked RIG-I-induced ISRE activation after intracellular (IC) poly (I:C)-LMW stimulation (5 μg/mL, the same with other experiments shown below) (Fig. 1C)
Summary
RIG-I, melanoma differentiation factor 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) belong to the DExD/H-box family of helicases[2]. Ubiquitination plays crucial roles in RIG-I activation as well as the type I IFN signaling pathway[6]. RIG-I-2CARD can be ubiquitinated at lysines (K) 48, 99, 154, 164, 169, 172, 181, 190 and 193 Among these sites, K164 and K172 are most important ubiquitination sites for RIG-I-2CARD to activate type I IFN signaling[7,11,12]. In this study, we sought to determine the mechanism by which the regulation of RIG-I ubiquitination by anchored and unanchored ubiquitin chains is involved in antiviral immune response. We examined the functional implications of multi-site/type ubiquitination of RIG-I in the innate immune response
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