Regulation of Necroptosis and the Type Interferon Response by Monkeypox/mpox Virus.

Monkeypox/mpox virus (MPXV) has re-emerged as the most important orthopoxvirus infection of humans. Despite being pathogenic, it contains a natural truncation at the N-terminus of its homologue of the vaccinia virus (VACV) innate immune evasion protein, E3, leading to the loss of the first 37 amino acids. Our previous data have shown that VACV E3 protein is required for interferon-resistance of VACV. The N-terminal Z-nucleic acid binding domain is necessary to inhibit induction of necroptosis, as VACV containing an N-terminal deletion (VACV-E3L{Delta}37N) undergoes rapid ZBP1-dependent necroptotic cell death, through activation of RIPK3, and subsequent phosphorylation and trimerization of the executioner of necroptosis, MLKL. Despite lacking parts of the N-terminus, MPXV has evolved ways in circumventing necroptotic cell death in mouse L929 cells. Our data shows that MPXV infection inhibits phosphorylation of MLKL and does not lead to MLKL trimerization, nor cell death. We show that MPXV inhibits necroptosis in two steps: by degrading RIPK3 through either a caspase-dependent pathway or through the "viral inducer of RIPK3 degradation" (vIRD) and by inhibiting MLKL aggregation. Additionally, MPXV shows interferon sensitivity that is fully ZBP1- and RIPK3- and MLKL-dependent, but independent of necroptosis. Addition of a pancaspase inhibitor (zVAD), or a proteasome inhibitor (MLN4924) does not increase cell death but leads to an increase in interferon sensitivity comparable to VACV-E3L{Delta}37N. Thus, treatment with an interferon inducer along with a pancaspase or proteasome inhibitor could potentially be a beneficial treatment against MPXV infections.