Porcine Epidemic Diarrhea Virus Infection Induces Caspase-8-Mediated G3BP1 Cleavage and Subverts Stress Granules To Promote Viral Replication

Abstract

Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus causing severe diarrhea and high mortality rates in suckling piglets and posing significant economic impact. PEDV replication is completed and results in a large amount of RNA in the cytoplasm. Stress granules (SGs) are dynamic cytosolic RNA granules formed under various stress conditions, including viral infections. Several previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. However, the underlying mechanisms are poorly understood. This study aimed to delineate the molecular mechanisms regulating the SG response to PEDV infection. SG formation is induced early during PEDV infection, but as infection proceeds, this ability is lost and SGs disappear at late stages of infection (>18 h postinfection). PEDV infection resulted in the cleavage of Ras-GTPase-activating protein-binding protein 1 (G3BP1) mediated by caspase-8. Using mutational analysis, the PEDV-induced cleavage site within G3BP1 was identified, which differed from the 3C protease cleavage site previously identified. Furthermore, G3BP1 cleavage by caspase-8 at D168 and D169 was confirmed in vitro as well as in vivo The overexpression of cleavage-resistant G3BP1 conferred persistent SG formation and suppression of viral replication. Additionally, the knockdown of endogenous G3BP1 abolished SG formation and potentiated viral replication. Taken together, these data provide new insights into novel strategies in which PEDV limits the host stress response and antiviral responses and indicate that caspase-8-mediated G3BP1 cleavage is important in the failure of host defense against PEDV infection.IMPORTANCE Coronaviruses (CoVs) are drawing extensive attention again since the outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. CoVs are prone to variation and own the transmission capability by crossing the species barrier resulting in reemergence. How CoVs manipulate the antiviral responses of their hosts needs to be explored. Overall, the study provides new insight into how porcine epidemic diarrhea virus (PEDV) impaired SG assembly by targeting G3BP1 via the host proteinase caspase-8. These findings enhanced the understanding of PEDV infection and might help identify new antiviral targets that could inhibit viral replication and limit the pathogenesis of PEDV.

Keywords: G3BP1; PEDV; caspase-8; stress granules; viral replication.

FIG 1

SGs were induced transiently upon PEDV infection. (A) Vero E6 cells were transfected with poly(I:C) (2 μg/ml) for 12 h and mock infected or infected with SQ2014 (1 MOI) for 6, 12, 18, 24, and 36 h. The cells were fixed and stained with G3BP1 antibody (A302-033A) and Alexa 488-conjugated goat anti-mouse IgG antibodies and then stained with PEDV-N antibody and Alexa 555-conjugated goat anti-rabbit IgG antibody. The nuclei were stained with DAPI. The images were acquired with a Nikon confocal microscope. (B) Graph shows the percentage of SG formation in PEDV-infected cells (about 200 cells). The means and standard deviations of three independent experimental replicates are shown. The results were analyzed using the Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

The replication of PEDV was inhibited by SG formation. A total of 5 × 10⁵ cells were seeded in each well of six-well plates after 18 h and transfected with pEGFP-G3BP1 or empty vector for 24 h, followed by SQ2014 infection (0.01 MOI) for 24 h. N, G3BP1, and actin were detected by using RT-PCR (A) and immunoblotting (B), and virus titers were measured by plaque formation assay (E). A total of 3 × 10⁵ cells were seeded in each well of six-well plates after 18 h and transfected with nontargeting scrambled siRNA or targeting G3BP1 siRNA for 48 h, followed by infection with SQ2014 (0.01 MOI) for 24 h. N, G3BP1, and actin were detected by using RT-PCR (C) and immunoblotting (D), and virus titers were measured by plaque formation assay (F). Con, control.

FIG 3

G3BP1 was cleaved in the late stage of PEDV infection. (A) Vero E6 cells were mock infected or infected with SQ2014 (1 MOI) for 6, 12, 18, 24, and 36 h. Whole-cell extracts were prepared and the protein levels of G3BP1, PEDV-N, and actin were analyzed by immunoblotting. The C-terminal cleavage products of G3BP1 (designated G3BP1_cpc) are presented with arrows. (B) Vero E6 cells were mock infected or infected with three different strains of PEDV (CV777, HLJBY, and SQ2014) at 1 MOI for 24 or 36 h. Whole-cell extracts were prepared, and the protein level of G3BP1 was analyzed using immunoblotting with the G3BP1 antibody (A302-034A). (C) Schematic presentation for recombinant G3BP1 and G3BP1 antibody epitope (A302-034A). (D) Vero E6 cells were transfected with pcDNA3-2×Flag-G3BP1-HA (1 μg) for 24 h, and then the cells were mock infected or infected with SQ2014 (1 MOI) for 24 h and 36 h. Whole-cell lysates were prepared and analyzed by immunoblotting. G3BP1 and the N-terminal cleavage products of G3BP1 (designated G3BP1_cpn) were detected by Flag antibody. G3BP1 and G3BP1_cpc were detected using HA and G3BP1 antibody (A302-034A). The arrows denote the positions of all G3BP1 bands. (E) Schematic presentation of amino acid sequence alignment of G3BP1, G3BP2a, and G3BP2b (performed using Clustal Omega [https://www.ebi.ac.uk/Tools/msa/clustalo/]). Sequences and NCBI accession numbers used in this alignment were as follows: G3BP1 (NP_005745.1), G3BP2a (NP_036429.2), and G3BP2b (NP_987100.1). (F) Vero E6 cells were mock infected or infected with SQ2014 (1 MOI) for 12, 24, and 36 h. Whole-cell extracts were prepared and analyzed by immunoblotting. G3BP2a and G3BP2b were detected using the G3BP2 antibody. Asterisks indicate positions which have a single, fully conserved residue. A colon indicates conservation between groups of strongly similar properties. A period indicates conservation between groups of weakly similar properties.

FIG 4

G3BP1 was cleaved by caspase-8. (A) Vero E6 cells were mock infected or infected with SQ2014 (1 MOI) for 6, 12, 18, 24, and 36 h. Whole-cell extracts were prepared and analyzed by immunoblotting with the indicated protein antibodies. (B) Vero E6 cells were treated with the caspase-8 inhibitor Z-IETD-fmk (10, 20, and 50 μM) or an equivalent volume of dimethyl sulfoxide (DMSO) for 1 h, and then the cells were mock infected or infected with SQ2014 (1 MOI) for 36 h. Whole-cell extracts were prepared and analyzed by immunoblotting with the indicated protein antibodies. (C) Vero E6 cells were transfected with pcDNA4-G3BP1-2×Flag (1 μg), along with increasing quantities of pcDNA4-caspase-8-HA (0, 25, 75, and 125 ng) for 24 h. Whole-cell lysates were prepared and analyzed by immunoblotting. G3BP1 was detected by the Flag antibody, and caspase-8 was detected by the HA antibody. Purified recombinant His-tagged G3BP1 was incubated with or without the purified recombinant caspase-8 (0, 1, and 10 μg) at 37°C for 4 h, and the reaction products were analyzed by SDS-PAGE (D) or immunoblotting with the G3BP1 antibody (E). The positions of N-terminal cleavage products of G3BP1 (designated G3BP1_cpn) and C-terminal cleavage products of G3BP1 (designated G3BP1_cpc) are indicated by arrows.

FIG 5

G3BP1 was cleaved by caspase-8 at Asp168 and Asp169. (A) Schematic presentation of G3BP1 mutations. Purified wild-type or mutants of His-tagged G3BP1 were incubated with or without the purified recombinant caspase-8 (10 μg) at 37°C for 4 h, and the products were analyzed by SDS-PAGE (B) or immunoblotting with the G3BP1 antibody (C). (D) Vero E6 cells were transfected with wild-type or mutants of EGFP-tagged G3BP1 for 24 h and then mock infected or infected with SQ2014 (1 MOI) for 24 h. G3BP1, PEDV-N, and actin were detected by immunoblotting. (E) Schematic presentation of G3BP1 cleavage sites. Porcine G3BP1 (E284) corresponds to the human G3BP1 (E285).

FIG 6

SGs were rescued in Vero E6 cells expressing cleavage-resistant G3BP1. (A) Vero E6 cells were transfected with WT (1 μg), M3 (1.2 μg), M4 (1.2 μg), and M7 (1.2 μg) for 24 h and then mock infected or infected with SQ2014 (1 MOI) for 24 h. Then, the cells were fixed and stained with PEDV-N antibody and Alexa 555-conjugated goat anti-rabbit IgG antibody (red). The nuclei were stained with DAPI (blue). Images were acquired with a Nikon immunofluorescence microscope. (B) Graph shows the percentage of cells containing SGs (counting 200 cells). The means and standard deviations of three independent experimental replicates are shown. (C) Vero E6 cells were treated with Z-VAD-fmk (20 μM), Z-IETD-fmk (20 μM). or an equivalent volume of dimethyl sulfoxide (DMSO) for 1 h, and then the cells were infected with SQ2014 (1 MOI) for 0, 24, and 36 h. The cells were fixed and stained with G3BP1 antibody (A302-033A) and PEDV-N antibody. The nuclei were stained with DAPI. The images were acquired with a Nikon confocal microscope. (D) Graph shows the percentage of SG formation in PEDV-infected cells (about 200 cells). The means and standard deviations of three independent experimental replicates are shown.

FIG 7

Replication of PEDV was inhibited by cleavage-resistant G3BP1. Vero E6 cells were transfected with WT (1 μg), M3 (1.2 μg), M4 (1.2 μg), and M7 (1.2 μg) for 24 h and then infected with SQ2014 (0.01 MOI) for 24 h. PEDV-N, G3BP1, and actin were detected by semiquantitative RT-PCR (A) and immunoblotting (B), and virus titers were detected by plaque formation assay (C). ***, P < 0.001. (D) Vero E6 cells were treated with Z-VAD-fmk (20 μM), Z-IETD-fmk (20 μM), or an equivalent volume of dimethyl sulfoxide (DMSO) for 1 h, and then the cells were infected with SQ2014 (1 MOI) for 0, 24, and 36 h. Whole-cell extracts were prepared and analyzed by immunoblotting with the indicated antibodies.

FIG 8

Model for the interplay between caspase-8 and G3BP1-containing SGs during PEDV infection. The host cells sensed the infection of PEDV and induced G3BP1 dimerization/oligomerization and SG formation. PEDV infection activated caspase-8 and subsequently led to G3BP1 cleavage to counteract the inhibitory effect of SGs. PEDV infection induced apoptosis through caspase-8 activation.