Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression

Abstract

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/β) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (L pro) of footand-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/β gene transcription; however, the exact mechanism is unknown. The proteolytic activity of L pro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, L pro has been demonstrated to have deubiquitination/deISGylation activity. L pro 's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/β gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by L pro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing L pro. In vitro cleavage experiments revealed that L pro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-L pro , but only observed decreasing levels of

Virus–Host Interactions in Foot-and-Mouth Disease Virus Infection

“…The viruses infect and damage the cells that express IL-2, IL-12, IL-15, IL-18, and IFNs which are required for NK cells activation. L pro blocks the activation of RLR pathway and inhibits the expression of various cytokines (89,90). Meanwhile, L pro cleaves eIF4G which also decreases the expression of IFNs and cytokines.…”

Use of Protein Pegylation to Prolong the Antiviral Effect of IFN Against FMDV

“…Two other genes that are part of the cellular ISGylation machinery, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18, were also upregulated in response to PEGpoIFNα. It has recently been demonstrated that FMDV actively induces protein deISGylation ( Medina et al, 2020a ; Visser et al, 2020 ). Furthermore, overexpression of ISG15 and the ISGylation machinery results in moderate inhibition of FMDV replication in porcine cells ( Medina et al, 2020a ).…”

Uncovering targets of the Leader protease: Linking RNA‐mediated pathways and antiviral defense

“…Relevant proteins of this pathway (RIG‐I, MAVS, TBK1, IRF‐3) are regulated by ubiquitin‐dependent modifications. In this respect, the TBKI kinase, responsible for phosphorylation of the transcription factor IRF3 is also cleaved by L pro in FMDV‐infected cells (Visser et al, 2020) at a KKLK site, resembling the minimal motif defined in Daxx and Gemin5 (Figure 2c). Interestingly, the mutations in L pro that impaired the reduction of IFN‐β mRNA levels displayed cleavage defects and/or degradation of the signaling proteins MAVS, TBK1, and NF‐κB p65, establishing a direct link between L pro activity and IFN reduction.…”

“…Interestingly, the mutations in L pro that impaired the reduction of IFN-β mRNA levels displayed cleavage defects and/or degradation of the signaling proteins MAVS, TBK1, and NF-κB p65, establishing a direct link between L pro activity and IFN reduction. Thus, L pro 's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with suppressing IFN-α/β gene transcription (Medina et al, 2020;Visser et al, 2020).…”