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 RNA-dependent protein kinase (PKR) or eIF2α, 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.

Keywords: Aphthovirus; FMDV; G3BP1; G3BP2; SGs; Stress granules.

FIG 1

FDMV suppresses SG formation. (A) LFPK αvβ6 cells were infected with FMDV-A12 and fixed at 4 and 6 hpi, and immunofluorescence staining was performed for the viral capsid protein VP1 (green) and the SG marker G3BP1 (red). (B) LFPK αvβ6 cells were treated with 500 μM sodium arsenite (NaArs) for 30 min to induce SG formation (as previously described [24, 26]), which were subsequently visualized by immunofluorescence staining for the SG marker G3BP1 (red). (C) LFPK αvβ6 cells were infected with FMDV-A12, treated with 500 μM sodium arsenite for the last 30 min to induce SG formation, and fixed at 4 hpi. Immunofluorescence staining was performed for the viral capsid protein VP1 (green) and the SG marker G3BP1 (red). (D) Similar to the image shown in panel C but with IBRS-2 cells.

FIG 2

Aphthovirus L^pro suppresses SG formation. (A) Schematic representation of viral genome of chimeric EMCV viruses that were constructed for this study. The endogenous stress response antagonist (leader) was inactivated by point mutations in its Zn finger domain (C19A/C22A), and subsequently, the genes encoding L^pro and 3C^pro were introduced at the 5′ end of the EMCV open reading frame. (B) HeLa R19 cells were infected at an MOI of 10 with the indicated chimeric EMCVs, and subsequently, virus growth kinetics were determined using RT-qPCR analysis with EMCV-specific primers. (C) HeLa R19 cells were infected at an MOI of 10 with the indicated chimeric EMCVs and fixed at 6 hpi. Subsequent immunofluorescence staining was performed against the SG markers G3BP1 (green), TIA-1 (red), and G3BP2 (cyan). (D) Quantification of data from panel C in which the numbers of SGs and SG sizes were analyzed for at least 50 cells per condition. One-way ANOVA with the Bonferroni post hoc test was used to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Effect of L^pro on SG formation is dependent on the catalytic activity of L^pro. (A) HeLa R19 cells were infected at an MOI of 10 with chimeric EMCV expressing L^pro (EMCV-FL) or a catalytically inactive L^pro (EMCV-FL C51A) and fixed at 6 hpi. SG formation was visualized by immunofluorescence staining for the SG markers G3BP1 (green), eIF3 (red), and G3BP2 (cyan). (B) Quantification of data from panel A in which SG sizes were analyzed for at least 50 cells per condition. One-way ANOVA with the Bonferroni post hoc test was used to determine statistical significance. *, P < 0.05.

FIG 4

Leaderless FMDV is unable to suppress SG formation. (A) LFPK αvβ6 cells were infected with FMDV-A12 or leaderless FMDV-A12 (LLV) and fixed at 4 hpi, and immunofluorescence staining was performed for the viral capsid protein VP1 (green) and the SG marker G3BP1 (red). (B) Quantification of data from panel A in which the percentages of SG-positive infected cells and the numbers of SGs per cell were determined in at least 50 cells. A one-tailed t test was used to determine statistical significance. **, P < 0.01; ***, P < 0.001.

FIG 5

L^pro does not inhibit PKR signaling. HeLa R19 cells were infected at an MOI of 10 with EMCV or EMCV-L^Zn (A) or the indicated chimeric EMCVs (B) and fixed at 6 hpi. Subsequent flow cytometry staining was performed for dsRNA and p-PKR or for dsRNA and p-eIF2α. Graphs represent the levels of p-PKR or p-eIF2α in dsRNA-positive (infected) cells. Dashed lines indicate the levels of p-PKR and p-eIF2α in EMCV-L^Zn infected cells.

FIG 6

SG scaffold proteins are cleaved during aphthovirus infection. (A) LFPK αvβ6 cells were infected with FMDV-A12 or leaderless FMDV-A12 (LLV), and cells were lysed at the indicated times postinfection. Western blot analyses were performed for G3BP1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the viral proteins VP1 and L^pro. (B) HeLa R19 cells were infected at an MOI of 10 with CVB3 or ERAV, and the cells were lysed at the indicated times postinfection. Western blot analyses were performed for SG proteins G3BP1, G3BP2, and TIA-1. (C) HeLa R19 cells were infected with ERAV at an MOI of 10 or treated with 5 μg/ml actinomycin D. Subsequently, the cells were incubated for 16 h in the presence or absence of 10 μM Q-VD. Cell lysates were subjected to Western blot analyses for G3BP1 and G3BP2, translation initiation factor 4G (eIF4G), and PARP. Arrows indicate full-length proteins, black arrowheads indicate aphthovirus-induced cleavage products, and gray arrowheads indicate cleavage products induced upon CVB3 infection (B) or cleavage products induced upon the induction of apoptosis (C).

FIG 7

Aphthovirus L^pro and 3C^pro cleave SG proteins. (A) HeLa R19 cells were infected at an MOI of 10 with EMCV, EMCV-L^Zn, or CVB3, and the cells were lysed at the indicated times postinfection. Western blot analyses were performed with two different antibodies against G3BP1 (recognizing different epitopes) and G3BP2. (B) HeLa R19 cells were infected at an MOI of 10 with the indicated chimeric EMCVs or ERAV, and the cells were lysed at 8 hpi (ECMVs) or 10 hpi (ERAV). Subsequent Western blot analyses were performed for G3BP1 and G3BP2. *, additional (artificial) G3BP2 cleavage product. (C) HeLa R19 cells were infected at an MOI of 10 with EMCV-FL or EMCV-FL C51A, and the cells were lysed at the indicated time points. Subsequent Western blot analyses were performed for G3BP1 and G3BP2. (D and E) HEK293T cells were cotransfected with a Flag-tagged G3BP1 or G3BP2 and an EGFP-tagged viral protease, as indicated. At 16 h posttransfection, the cells were lysed and subjected to Western blot analyses for FLAG and EGFP. Arrows indicate full-length proteins, black arrowheads indicate L^pro-induced cleavage products, and the gray arrowhead indicates 3C^pro-induced cleavage products. (F) Schematic representation of approximate cleavage sites, based on the apparent molecular weights of the cleavage products, in G3BP1 and G3BP2 for the different aphthovirus proteases. ■, region in G3BP2 containing multiple FMDV L^pro cleavage sites.