Cleavage of 14-3-3ε by the enteroviral 3C protease dampens RIG-I mediated antiviral signaling

Abstract

Viruses have evolved diverse strategies to evade the host innate immune response and promote infection. The RIG-I-like-receptors RIG-I and MDA5 (RLRs) are antiviral factors that sense viral RNA and signal downstream via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon (IFN) expression. 14-3-3ϵ is a key component of the RIG-I translocon complex that interacts with MAVS at the mitochondrial membrane; however, the exact role of 14-3-3ϵ in this pathway is not well understood. In this study, we demonstrate that 14-3-3ϵ is a direct substrate of both the poliovirus and coxsackievirus B3 (CVB3) 3C proteases (3C pro ), and that it is cleaved at Q236 ¯ G237, resulting in the generation of N- and C-terminal fragments of 27.0 and 2.1 kDa, respectively. Expression of the N-terminal cleavage fragment in cells reduces IFNB mRNA production during poly(I:C) stimulation, thus suggesting an antagonistic effect in the presence of the endogenous 14-3-3ϵ protein. The N-terminal 14-3-3ϵ fragment does not interact with RIG-I in co-immunoprecipitation assays, nor can it facilitate RIG-I translocation to the mitochondria. Probing the intrinsically disordered C-terminal region identifies key residues responsible for RIG-I signaling. Finally, overexpression of the N-terminal fragment promotes CVB3 infection and influenza A virus (H1N1) RNA production and reduces IFNB mRNA production during infection. The strategic enterovirus 3C pro -mediated cleavage of 14-3-3ϵ antagonizes RIG-I signaling by disrupting critical interactions within the RIG-I translocon complex, thus contributing to evasion of the host antiviral response.

Encephalomyocarditis virus protein 2B* antagonises innate immune signalling by interacting with 14-3-3 protein family members

“…A short linear motif at the C terminus of 2B* is essential for 14-3-3 binding. Interaction of 2B* with the 14-3-3 members reduced transcription of both IFNB1 and IL6 in addition to interferon stimulated gene (ISG) transcripts, in accordance with recently published results describing a role of 14-3-3 proteins in promoting antiviral immunity (7)(8)(9)(10). This antagonism of the innate immune system, via sequestration of the 14-3-3 family, is distinct from the previously described role of 2B* in promoting plaque size, thus indicating that 2B* possesses multiple functions which contribute to efficient EMCV replication and transmission.…”

“…Presumably, the 2B*:14-3-3 interaction performs an additional function that is beneficial to the virus. We hypothesised that the sequestration of 14-3-3 proteins by 2B* may contribute to the evasion of innate immune signalling as other viral binding partners of 14-3-3 proteins -such as the 3C protease of enterovirus -are known to have similar effects (7)(8)(9)(10). During viral infection, RIG-I is relocalised from the cytosol to the mitochondria by 14-3-3ε which forms a complex with both RIG-I and TRIM25 (20), the ubiquitin ligase essential for the antiviral function of RIG-I (26), enabling the activation of MAVS on the mitochondrial membrane.…”