A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses

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.


Figure S1
Experimental data used for parameter estimation Text S1 Model formulation for random mechanism Text S2 Model formulation for sequential mechanism 1 Text S3 Model formulation for sequential mechanism 2 Text S4 Model formulation for sequential mechanism 3 Text S5 Model formulation for hierarchical mechanism 1 Text S6 Model formulation for hierarchical mechanism 2 Text S7 Model formulation for hierarchical mechanism 3 Text S8 Optimization algorithm Text S9 Parameter sensitivity analysis Table S1 Quantification of robustness indexes of 7 models  !"#"$%&'() !" ub !"# !"# RIG − I !" −unanc ub RIGIuc unanc . The degradation rates of these tetramers were assumed to be the same ( 2 d ).

ODEs of RIG-I downstream pathway in random mechanism
The regulations involved in the RIG-I downstream pathway MAVS-pIRF3-ISRE were modeled using Hill functions as follows: : : Text S2 Model formulation for sequential mechanism 1 2.1 Biochemical reactions of RIG-I ubiquitination in sequential mechanism 1 [ ]

Rate equations of RIG-I ubiquitination in sequential mechanism 1
Text S3 Model formulation for sequential mechanism 2

Biochemical reactions of RIG-I ubiquitination in sequential mechanism 2
Reactions R 1 -R 4 and R 9 -R 14 were same with that in Text S2.1. The following are reactions different from that in sequential mechanism 1.

Rate equations of RIG-I ubiquitination in sequential mechanism 2
Rate equations 1 V -4 V and 9 V -14 V were same with that in Text S2.2. The following are rate equations different from that in sequential mechanism 1.

ODEs model of RIG-I ubiquitination in sequential mechanism 2
Same with that in Text S2.3 in the form

ODEs model of RIG-I downstream pathway in sequential mechanism 2
The activation of MAVS was modeled as follows: Text S4 Model formulation for sequential mechanism 3

Biochemical reactions of RIG-I ubiquitination in sequential mechanism 3
Reactions R 1 -R 2 and R 9 -R 14 were same with that in Text S2.1. The following are reactions different from that in sequential mechanism 1.

Rate equations of RIG-I ubiquitination in sequential mechanism 3
Rate equations 1 V -2 V and 9 V -14 V were same with that in Text S2.

ODEs model of RIG-I ubiquitination in sequential mechanism 3
Same with that in Text S2.3 in the form

ODEs model of RIG-I downstream pathway in sequential mechanism 3
The activation of MAVS was modeled as follows: Text S5 Model formulation for hierarchical mechanism 1

Rate equations of RIG-I ubiquitination in hierarchical mechanism
Text S6 Model formulation for hierarchical mechanism 2

Biochemical reactions of RIG-I ubiquitination in hierarchical mechanism 2
Reactions R 1 -R 2 and R 12 -R 19 were same with that in Text S5.1. The following are reactions different from that in sequential mechanism 1.

ODEs model of RIG-I ubiquitination in hierarchical mechanism 2
[ ] ub RIGIuc unanc were same with that in Text S5.3.

ODEs model of RIG-I downstream pathway in hierarchical mechanism 2
The activation of MAVS was modeled as follows: Text S7 Model formulation for hierarchical mechanism 3

Biochemical reactions of RIG-I ubiquitination in hierarchical mechanism 3
Same with that in hierarchical mechanism 1 (Text S5).

Rate equations of RIG-I ubiquitination in hierarchical mechanism 3
Same with that in hierarchical mechanism 1 (Text S5).

ODEs model of RIG-I ubiquitination in hierarchical mechanism 3
Same with that in hierarchical mechanism 1 (Text S5).

ODEs model of RIG-I downstream pathway in hierarchical mechanism 3
The activation of MAVS was modeled as follows: