Foot-and-Mouth Disease Virus Leader Protease Cleaves G3BP1 and G3BP2 and Inhibits Stress Granule Formation

Abstract

Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV; genus Aphthovirus), one of the most notorious pathogens in the global livestock industry, needs to navigate antiviral host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I interferon (IFN) response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV leader protease (L pro ) or 3C pro , we demonstrate an essential role for L pro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking L pro resulted in SG formation. Additionally, we show that L pro cleaves the known SG scaffold proteins G3BP1 and G3BP2 but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) L pro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved among aphthoviruses. Neither FMDV nor ERAV L pro interfered with phosphorylation of RNAdependent protein kinase (PKR) or eIF2␣, indicating that L pro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response. IMPORTANCE The picornavirus foot-and-mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on the global livestock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral double-stranded RNA (dsRNA) is detected, which results in the activation of the RNA-dependent protein kinase (PKR)-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here, we show that FMDV can suppress SG formation via its leader protease (L pro ). Simultaneously, we observed that L pro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of the antiviral host response evasion strategies of FMDV may help to develop countermeasures to control FMDV infections in the future.

Essential Role of Enterovirus 2A Protease in Counteracting Stress Granule Formation and the Induction of Type I Interferon

“…Indeed, infection with EMCV-2A pro , but not EMCV-2Am, resulted in cleavage of eIF4G, while infection with EMCV-3C pro resulted in G3BP1 cleavage. No G3BP1 cleavage was observed in cells infected with either EMCV-L Zn , which is consistent with our previous observations (11,41,42), or EMCV-3Cm.…”

“…It has been suggested that EMCV 3C pro cleaves G3BP1 and that this prevents SG assembly, although this cleavage is observed only at very late stages of infection (10 to 12 hpi) (40). In contrast, we and others did not observe G3BP1 cleavage in cells infected with either EMCV or Theiler's murine encephalomyelitis virus, and instead provided extensive evidence that the leader protein has an essential role in suppressing SG formation in cardiovirus-infected cells (11,41,42). As part of these studies, we generated EMCV-L Zn , a mutant virus in which the leader protein is inactivated by mutations that disrupt its zinc finger domain and that, as a consequence, can no longer suppress SG formation.…”

“…The oligonucleotides used for these PCRs encode flanking XhoI and NotI restriction sites that were used to ligate the PCR products into the desired plasmids. The 3C pro genes were ligated into the pcDNA-GFP vector, while the 2A pro genes were ligated into the pIRES-EGFP-MCS plasmid, both of which plasmids were described previously (42).…”

Innate Immune Evasion by Human Respiratory RNA Viruses

“…For rhinoviruses, nothing is known about their capacity to manipulate stress granule formation, however, for other picornaviruses the 2A and L proteases have recently been shown to interfere by cleaving stress granule factors such as G3BP1 and G3BP2 [106][107][108][109]. A recent report showed that binding of eiF4GI translation factor to stress granule-inducing protein G3BP1 is essential for antiviral stress granule formation, and this interaction is disrupted by the 2A or L proteases of picornaviruses [110].…”

Foot-and-Mouth Disease Virus 3A Protein Causes Upregulation of Autophagy-Related Protein LRRC25 To Inhibit the G3BP1-Mediated RIG-Like Helicase-Signaling Pathway

“…G3BP1, also known as G3BP or HDH-VIII, is a ubiquitously expressed protein and functions as a sequence-specific, phosphorylation-dependent helicase, a cofactor, an endoribonuclease, and more (19). Recently, it has been reported that FMDV L pro and 3C pro may cleave G3BP1 to inhibit stress granule (SG) formation; however, FMDV L pro and 3C pro do not interact with G3BP1 (20,21). This finding prompted us to determine the FMDV proteins that interact with G3BP1 and the mechanism by which it regulates G3BP1 to facilitate FMDV replication and growth.…”