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

2B* is a poorly characterized protein encoded by an overlapping ORF in the genome of encephalomyocarditis virus (EMCV). We have previously found 2B* to have a role in innate immune antagonism; however, this role is distinct from an earlier described phenotype whereby 2B*KO viruses exhibit extremely small plaques compared to WT. Here, we report that the small plaque phenotype is recapitulated by novel EMCV mutant viruses harbouring mutations across the C-terminal domain of 2B*, confirming a functional role of 2B* in promoting viral spread. We found that 2B*KO EMCV displays impaired extracellular virus titres compared to WT EMCV, despite producing a similar number of infectious particles overall. This correlates with a reduction in cell lysis and lower levels of caspase-3 cleavage occurring during infection. Further investigation using caspase inhibitors and knockout cells revealed that WT EMCV can utilize both caspase-3-dependent and caspase-3-independent pathways to achieve cell lysis, the former of which is likely to be GSDME-mediated pyroptosis. 2B* increases the efficiency of both lytic pathways through an as-yet-undefined mechanism. This work reveals 2B*, a protein only found in EMCV, to be a key regulator of multiple lytic cell death pathways, leading to enhanced rates of virus release. This explains the rapid cell death observed during WT EMCV infection and the small plaque phenotype seen in both 2B*KO and previously described 2B* mutant viruses.