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 retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and MDA5 are antiviral factors that sense viral RNA and trigger downstream signal via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon 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. While the exogenous expression of wild-type 14-3-3ε enhances IFNB mRNA production during poly(I:C) stimulation, expression of the truncated N-terminal fragment does not. 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 the interaction between 14-3-3ε and RIG-I. Finally, overexpression of the N-terminal fragment promotes CVB3 infection in mammalian cells. 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. IMPORTANCE Host antiviral factors work to sense virus infection through various mechanisms, including a complex signaling pathway known as the retinoic acid-inducible gene I (RIG-I)-like receptor pathway. This pathway drives the production of antiviral molecules known as interferons, which are necessary to establish an antiviral state in the cellular environment. Key to this antiviral signaling pathway is the small chaperone protein 14-3-3ε, which facilitates the delivery of a viral sensor protein, RIG-I, to the mitochondria. In this study, we show that the enteroviral 3C protease cleaves 14-3-3ε during infection, rendering it incapable of facilitating this antiviral response. We also find that the resulting N-terminal cleavage fragment dampens RIG-I signaling and promotes virus infection. Our findings reveal a novel viral strategy that restricts the antiviral host response and provides insights into the mechanisms underlying 14-3-3ε function in RIG-I antiviral signaling.

Temporal regulation of MDA5 inactivation by Caspase-3 dependent cleavage of 14-3-3η

“…In previous reports, 14-3-3ε had decreased binding affinity to its target protein, Bad, after Caspase-3 cleaved 14-3-3ε at Asp238 and removed partial C-tail [ 28 ]. While preparing this report, a study revealed that EV71 3C protease cleaved 14-3-3ε at a site close to a known Caspase-3 cleavage site at the C-tail, and the cleavage impaired the ability of 14-3-3ε to interact with RIG-I [ 41 ]. It is fascinating to know whether it is a common regulatory method for cells to mediate cleavage on other 14-3-3 proteins by Caspase-3 after infection for limiting the overactivation of RLR-signaling.…”

The 2023 Report on the Proteome from the HUPO Human Proteome Project

“…To counteract such host responses, viruses have acquired a range of mechanisms for immune evasion. Demonstrating the value of understanding N-terminal protein processing, viral 3C proteases from poliovirus and coxsackievirus B3 were shown to target and cleave 14-3-3ε to inhibit its functions and prevent the activation of host antiviral RIG-I signaling . Cross-linking mass spectrometry and quantitative proteomics spatially resolved a protein interactome map of human cytomegalovirus virions, demonstrating the layer-specific incorporation of host proteins …”

“…Demonstrating the value of understanding N-terminal protein processing, viral 3C proteases from poliovirus and coxsackievirus B3 were shown to target and cleave 14-3-3ε to inhibit its functions and prevent the activation of host antiviral RIG-I signaling. 82 Cross-linking mass spectrometry and quantitative proteomics spatially resolved a protein interactome map of human cytomegalovirus virions, demonstrating the layer-specific incorporation of host proteins. 83 This year marked the first proteomic investigation of a virus microenvironment formed via cell-to-cell communication following a viral infection.…”