Maharaj NP, Wies E, Stoll A, Gack MU

Retinoic acid-inducible gene-I (RIG-I) is a key sensor for viral RNA in the cytosol and initiates a signaling cascade leading to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent ...

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signaling function is regulated by the dynamic balance between phosphorylation and TRIM25-induced K(63)-linked ubiquitination: While ubiquitination of RIG-I is critical for RIG-I ability to induce an antiviral IFN response, phosphorylation of RIG-I at S(8) or T(170) suppresses RIG-I signal transducing activity under normal conditions. Here, we not only further define the roles of S(8) and T(170) phosphorylation for controlling RIG-I activity, but also identify conventional PKC-α/β as important negative regulators of the RIG-I signaling pathway. Mutational analysis indicated that while phosphorylation of either S(8) or T(170) potently inhibits RIG-I downstream signaling, dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression as well as gene silencing and specific inhibitor treatment demonstrated that PKC-α/β are primary kinases responsible for RIG-I S(8) and T(170) phosphorylation. Co-Immunoprecipitation showed that PKC-α/β interact with RIG-I under normal conditions leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination and thereby RIG-I-mediated IFN induction. PKC-α/β double knockdown cells exhibited markedly decreased S(8)/T(170) phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-α/β-induced RIG-I phosphorylation is a critical regulatory mechanism for controlling RIG-I antiviral signal transduction under normal conditions.

Date: Nov. 23, 2011

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