The EMCV protein 2B* is required for efficient cell lysis via both caspase-3-dependent and -independent pathways during infection

Fig. 1.. The interlinked cell death pathways of apoptosis, pyroptosis and necroptosis. Oligomerized GSDMC, GSDMD, GSDME and MLKL form non-selective pores, allowing the release of all cellular contents, including the indicated ions and immune signalling molecules, leading to the lytic cell death pathways of pyroptosis or necroptosis, as indicated. The resulting K+ efflux is also capable of activating the NLRP3 inflammasome in macrophage cells, as indicated by the dashed arrows. Created in BioRender. Stewart, H. (2025) https://BioRender.com/o62m219.

Fig. 2.. 2B* mutant viruses reproduce the small plaque phenotype associated with 2B*KO EMCV. (a) Alignment of all unique C. rueckerti (Cardiovirus A) 2B* sequences available on GenBank (3 October 2022). Duplicate sequences have been discarded. aa are colour coded according to their physicochemical properties. Asterisks indicate completely conserved residues, whereas colons and dots indicate stronger and weaker conservation (based on identity and shared physicochemical properties). Regions targeted by mutation are boxed in cyan, blue or purple. (b) BSR cells were infected with the respective viruses for 1 h prior to being overlaid with a semi-solid medium (1% low melting point agarose) and incubated for 48 h. Data shown are the size distribution of 60 randomly chosen plaques sampled over three biological repeats. Dotted lines indicate the median, upper and lower quartiles of the plaque size for each mutant virus. Statistical analysis (ratio-paired t-test): ****P≤0.0001.

Fig. 3.. 2B*KO EMCV exhibits impaired extracellular virus titres in a variety of cell lines. Confluent monolayers of BHK-21 (a), HeLa (b), MEF (c) and RAW264.7 (d) cells were infected with either WT EMCV or 2B*KO EMCV at an m.o.i. of 5.0 as specified. At the indicated timepoint, the medium was removed and frozen separately from the cells. The intracellular and extracellular viral titre in each sample was measured by plaque assay and normalized by the number of cells infected at 0 hpi (viral p.f.u. in each fraction per 10 000 cells) (left-hand panels). Total virus titres were calculated from the measured intracellular and extracellular titres (right-hand panels). Data represent the mean±sem of two independent biological repeats.

Fig. 4.. 2B*KO EMCV exhibits impaired cell lysis relative to WT EMCV. Confluent monolayers of BHK-21 (a, b), HeLa (c), MEF (d) and RAW264.7 (e) cells were infected with either WT EMCV or 2B*KO EMCV at an m.o.i. of 5.0 or treated with STS (0.5 µM) (a only) as specified. The cytotoxicity in each sample was measured over time either using a luciferase-based assay (a) or an NIR object count (count) by live-cell imaging in triplicate wells using the Incucyte (Sartorius) live-cell microscope and analysis software and adjusted for cell confluence (b–e). Data shown are the mean±sd of four (a) or three (b–e) biological repeats, each using triplicate wells. Statistical analysis [ratio-paired t-test (a) and Student’s t-test (b–e)]: *P≤0.05, **P≤0.01, ***P≤0.001. Significance values are shown relative to WT EMCV.

Fig. 5.. 2B*KO EMCV-infected cells are morphologically distinct from both WT EMCV-infected and TMEV-infected MEF cells. MEF cells were either mock infected (a), treated with 1 µM STS (b) or infected at an m.o.i. of 5.0 with WT EMCV (c), 2B*KO EMCV (d) or TMEV (e). Electron micrographs show vacuoles (white arrowheads) in STS-treated cells, replication organelles (black arrows) in all infected samples and enlarged mitochondria with unusual round appearance (asterisks) in 2B*KO EMCV-infected cells. Electron lucent cytosol, indicating a loss of membrane permeability, is visible in WT EMCV-infected cells (right-hand panel). The WT EMCV-infected cells were harvested at 7 hpi; all other samples were harvested at 12 hpi. Images are two representative images of one sample, harvested at the same timepoint.

Fig. 6.. WT EMCV induces more cleavage of caspase-3 than 2B*KO EMCV at timepoints, which showed differences in cell lysis. BHK-21 cells were infected with WT EMCV or 2B*KO EMCV at an m.o.i. of 5.0, mock infected or treated with 0.5 µM STS. Cells were fixed at 8 hpi (a) or 14 hpi (b), permeabilized and stained with antibodies recognizing dsRNA (APC) or cleaved-caspase-3 (FITC). Samples were analysed using an LSR-II flow cytometer. Data shown are the indicated average (mean or mode) of cleaved caspase-3 in a minimum of three independent biological repeats. Statistical analysis (a, b, d) (Mann–Whitney U test) was used to compare the mean cleaved caspase-3 in the infected single cells, or the single cells in the mock-infected and STS-treated control samples. ns, not significant, *P≤0.05. (c) Histograms show the relative amount of dsRNA and cleaved caspase-3 in each cell in each sample of one biological repeat; data are representative of the three biological replicates. (d) The mode level of cleaved caspase-3 detected in each cell, in each sample, fixed at 14 hpi. Horizontal lines indicate the mean of the modes for each sample. Note that the effect from STS (the positive control) only becomes…

Fig. 7.. Efficient WT EMCV-induced cytotoxicity requires both caspase-3 and 2B*. Confluent monolayers of BHK-21 (a) or HeLa (b) cells were infected with either WT EMCV or 2B*KO EMCV at an m.o.i. of 5.0. The caspase-3/7 activity was measured in GCU per square micromolar by live-cell imaging in triplicate wells using the Incucyte (Sartorius) live-cell microscope and analysis software and adjusted for cell confluence. (c) Confluent monolayers of BHK-21 cells were infected with either WT EMCV or 2B*KO EMCV at an m.o.i. of 5.0 immediately prior to treatment with the pan-caspase inhibitor, zVAD-fmk. The cytotoxicity was measured in NIR object count (count) by live-cell imaging in triplicate wells using the Incucyte (Sartorius) live-cell microscope and analysis software and adjusted for cell confluence, also measured by the Incucyte. Data shown are the mean±sd of three biological repeats, each using triplicate wells. Statistical analysis (Student’s t-test): ns, not significant *P≤0.05, **P≤0.01. All statistical analyses compare the inhibitor-treated samples to the untreated sample infected with the same virus: orange asterisks represent the statistical comparison of cytotoxicity induced by…

Fig. 8.. Knockout of caspase-3 reduces both WT and 2B*KO EMCV-induced cell lysis. Monolayers of WT or caspase-3 KO HeLa cells were infected with either WT EMCV or 2B*KO EMCV at an m.o.i. of 5.0. Live-cell imaging was used to measure the cell death in each sample by NIR object count (count). NIR was analysed using the Incucyte live-cell imaging suite (Sartorius) and normalized to the cell confluence determined by the same software. The same data in (a) are represented in (b), following the removal of the WT HeLa cell samples for clarity. Data shown are the mean±sd of three biological repeats, each using triplicate wells. Statistical analysis (unpaired Welch t-test): *P≤0.05, **P≤0.01, ***P≤0.001. Statistical analyses (a) compare the cytotoxicity induced by the same virus in each cell line. Orange asterisks represent the statistical comparison of cytotoxicity induced by infection with WT EMCV, in WT HeLa cells compared to caspase-3 KO HeLa cells. Green asterisks indicate infection with 2B*KO EMCV, in WT HeLa cells compared to caspase-3 KO HeLa cells. (b) Statistical analyses compare the cytotoxicity induced by WT EMCV to 2B*KO EMCV in caspase-3 KO HeLa cells only (green asterisks). (c) The…

Fig. 9.. 2B* is required for efficient GSDME cleavage during EMCV infection. HEK293T cells were transiently transfected with pcDNA3.1-myc-GSDME-FLAG, or an uncleavable mutant GSDME (D270A), 24 h prior to infection with either WT EMCV or 2B*KO EMCV at an m.o.i. of 5.0. At the specified timepoints, 12 hpi (a) and 24 hpi (b), samples were frozen in a complete RIPA buffer. Proteins were resolved on two separate SDS-PAGE gels, followed by immunoblotting with the specified antibodies. Immunoblots shown are representative of two independent biological repeats. FL GSDME, full-length GSDME; C GSDME, C-terminal fragment of GSDME.

Fig. 10.. Inhibition of either palmitoylation or caspase-6 activation reduces WT EMCV-induced cell lysis. Monolayers of BHK-21 cells were infected with WT EMCV (a) or 2B*KO EMCV (b) at an m.o.i. of 5.0 prior to the addition of either the palmitoylation inhibitor 2-BP or the pan-caspase inhibitor zVAD-fmk. (c) Monolayers of BHK-21 cells were infected with WT EMCV at an m.o.i. of 5.0 prior to the addition of the specified inhibitor. Live-cell imaging was used to measure the cell death in each sample by NIR object count (count). NIR was analysed using the Incucyte live-cell imaging suite (Sartorius) and normalized to the cell confluence determined by the same software. Data shown are the mean±sd of three biological repeats, each using triplicate wells. Statistical analysis (Student’s t-test): ns, not significant, *P≤0.05, **P≤0.01. All statistical analyses compare the inhibitor-treated samples to the untreated sample.